Extended dicer substrate agents and methods for the specific inhibition of gene expression

ABSTRACT

The invention provides compositions and methods for reducing expression of a target gene in a cell, involving contacting a cell with an isolated double stranded nucleic acid (dsNA) in an amount effective to reduce expression of a target gene in a cell. The dsNAs of the invention possess a single stranded extension (in most embodiments, the single stranded extension comprises at least one modified nucleotide and/or phosphate back bone modification). Such single stranded extended Dicer-substrate siRNAs (DsiRNAs) were demonstrated to be effective RNA inhibitory agents compared to corresponding double stranded DsiRNAs.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 13/708,185, filed on Dec. 7, 2012 which is a divisional of U.S.patent application Ser. No. 12/824,011, filed on Jun. 25, 2010, whichissued as U.S. Pat. No. 8,349,809 on Jan. 8, 2014, which is acontinuation-in-part of U.S. patent application Ser. No. 12/704,256,filed on Feb. 11, 2010, which claims priority under 35 U.S.C. § 119(e)to U.S. provisional patent application No. 61/151,841, filed Feb. 11,2009; and U.S. Ser. No. 12/824,011 is also a continuation-in-part ofU.S. patent application Ser. No. 12/642,371, filed Dec. 18, 2009, whichissued as U.S. Pat. No. 8,513,207 on Aug. 20, 2013, which is related toand claims priority under 35 U.S.C. § 119(e) to the followingapplications: U.S. provisional patent application No. 61/138,946, filedDec. 18, 2008; U.S. provisional patent application No. 61/166,227, filedApr. 2, 2009; U.S. provisional patent application No. 61/173,505, filedApr. 28, 2009; U.S. provisional patenet application No. 61/173,514,filed Apr. 28, 2009; U.S. provisional patent application No. 61/173,521,filed Apr. 28, 2009; U.S. provisional patent application No. 61/173,525,filed Apr. 28, 2009; U.S. provisional patent application No. 61/173,532,filed Apr. 28, 2009; U.S. provisional patent application No. 61/173,538,filed Apr. 28, 2009; U.S. provisional patent application No. 61/173,544,filed Apr. 28, 2009; U.S. provisional patent application No. 61/173,549,filed Apr. 28, 2009; U.S. provisional patent application No. 61/173,554,filed Apr. 28, 2009; U.S. provisional patent application No. 61/173,556,filed Apr. 28, 2009; U.S. provisional patent application No. 61/173,558,filed Apr. 28, 2009; and U.S. provisional patent application No.61/173,563, filed Apr. 28, 2009. The entire teachings of the aboveapplications are incorporated herein by reference.

SEQUENCE LISTING

The attached sequence listing is hereby incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

Double-stranded RNA (dsRNA) agents possessing strand lengths of 25 to 35nucleotides have been described as effective inhibitors of target geneexpression in mammalian cells (Rossi et al., U.S. Patent PublicationNos. 2005/0244858 and 2005/0277610). dsRNA agents of such length arebelieved to be processed by the Dicer enzyme of the RNA interference(RNAi) pathway, leading such agents to be termed “Dicer substrate siRNA”(“DsiRNA”) agents. Certain modified structures of DsiRNA agents werepreviously described (Rossi et al., U.S. Patent Publication No.2007/0265220).

BRIEF SUMMARY OF THE INVENTION

The present invention is based, at least in part, upon the surprisingdiscovery that double stranded nucleic acid agents having strand lengthsin the range of 25-30 nucleotides in length that possess a singlestranded nucleotide region either at the 5′ terminus of the antisensestrand, at the 3′ terminus of the sense strand, or at the 5′ terminus ofthe sense strand are effective RNA interference agents. Inclusion of oneor more modified nucleotides and/or phosphate backbone modificationswithin the single stranded region of a single stranded extended DsiRNAcan impart certain advantages to such a modified DsiRNA molecule,including, e.g., enhanced efficacy (including enhanced potency and/orimproved duration of effect), display of a recognition domain forDNA-binding molecules, and other attributes associated with a singlestranded nucleotide region

Thus, in certain aspects, the instant invention provides RNA inhibitoryagents possessing enhanced efficacies at greater length (via moreprecise direction of the location of Dicer cleavage events) thanpreviously described RNA inhibitory agents, thereby allowing forgeneration of dsRNA-containing agents possessing enhanced efficacy,delivery, pharmacokinetic, pharmacodynamic and biodistributionattributes, as well as improved ability, e.g., to be successfullyformulated, to be targeted to a specific receptor, to be attached to anactive drug molecule and/or payload, to be attached to another activenucleic acid molecule, to be attached to a detection molecule, topossess (e.g., multiple) stabilizing modifications, etc.

In one aspect, the invention provides an isolated double strandednucleic acid having a first oligonucleotide strand having a 5′ terminusand a 3′ terminus and a second oligonucleotide strand having a 5′terminus and a 3′ terminus, where each 5′ terminus has a 5′ terminalnucleotide and each 3′ terminus has a 3′ terminal nucleotide, where thefirst strand is 25-30 nucleotide residues in length, where starting fromthe 5′ terminal nucleotide (position 1) positions 1 to 23 of the firststrand include at least 8 ribonucleotides; the second strand is 36-66nucleotide residues in length and, starting from the 3′ terminalnucleotide, includes at least 8 ribonucleotides in the positions pairedwith positions 1-23 of the first strand to form a duplex; where at leastthe 3′ terminal nucleotide, and up to 6 consecutive nucleotides 3′terminal of the second strand, is unpaired with the first strand,forming a 3′ single stranded overhang of 1-6 nucleotides; where at least10 consecutive nucleotides and at most 30 consecutive nucleotides, notincluding the unpaired 3′ terminal nucleotides of the second strand areunpaired with the first strand, thereby forming in the second strand a10-30 nucleotide single stranded 5′ overhang; where the 5′ terminal andthe 3′ terminal nucleotides of the first strand is each paired with acorresponding nucleotide of the second strand, the corresponding secondstrand nucleotide being consecutive to the second strand 3′ singlestranded overhang and the second strand 5′ overhang, respectively,thereby forming a substantially duplexed region between the first andsecond strands; and the second strand is sufficiently complementary to atarget RNA along at least 19 ribonucleotides of the second strand lengthto reduce target gene expression when the double stranded nucleic acidis introduced into a mammalian cell.

In another aspect the invention provides an isolated double strandednucleic acid having a first oligonucleotide strand having a 5′ terminusand a 3′ terminus and a second oligonucleotide strand having a 5′terminus and a 3′ terminus, where each 5′ terminus has a 5′ terminalnucleotide and each 3′ terminus has a 3′ terminal nucleotide, where thefirst strand is 35-60 nucleotide residues in length, where starting fromthe 5′ terminal nucleotide (position 1) positions 1 to 28 of the firststrand include at least 8 ribonucleotides; the second strand is 26-36nucleotide residues in length and, starting from the 3′ terminalnucleotide, includes at least 8 ribonucleotides in the positions pairedwith positions 1-23 of the first strand to form a duplex; where at leastthe 3′ terminal nucleotide, and up to 6 consecutive 3′ terminalnucleotides, of the second strand is unpaired with the first strand,forming a 3′ single stranded overhang of 1-6 nucleotides; where at least10 consecutive nucleotides and at most 30 consecutive nucleotides,including the 3′ terminal nucleotide of the first strand are unpairedwith the 5′ terminus of the second strand, thereby forming a 10-30nucleotide single stranded 3′ overhang; where the 5′ terminal nucleotideof the first strand is paired with the nucleotide of the second strandconsecutive to the second strand 3′ single stranded overhang, and the 5′terminal nucleotide of the second strand is paired with the nucleotideof the first strand consecutive to the first strand 3′ overhang, therebyforming a substantially duplexed region between the first and secondstrands; and the second strand is sufficiently complementary to a targetRNA along at least 19 ribonucleotides of the second strand length toreduce target gene expression when the double stranded nucleic acid isintroduced into a mammalian cell.

In another aspect the invention provides an isolated double strandednucleic acid having a first oligonucleotide strand having a 5′ terminusand a 3′ terminus and a second oligonucleotide strand having a 5′terminus and a 3′ terminus, where each 5′ terminus has a 5′ terminalnucleotide and each 3′ terminus has a 3′ terminal nucleotide, where thefirst strand is 35-66 nucleotide residues in length, where starting fromthe 5′ terminal nucleotide consecutive to the first strand 5′ singlestranded overhang (position 1^(F)) positions 1^(F) to 28^(F) of thefirst strand include at least 8 ribonucleotides; the second strand is25-36 nucleotide residues in length and, includes at least 8ribonucleotides in the positions paired with positions 1^(F)-23^(F) ofthe first strand to form a duplex; where the 3′ terminal nucleotide ofthe first strand and the 5′ terminal nucleotide of the second strandform a blunt end; where at least 10 consecutive nucleotides and at most30 consecutive nucleotides, including the 5′ terminal nucleotide of thefirst strand are unpaired with the 3′ terminus of the second strand,thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherethe 3′ terminal nucleotide of the second strand is paired with thenucleotide of the first strand consecutive to the first strand 5′ singlestranded overhang, and the 3′ terminal nucleotide of the first strand ispaired with the 5′ terminal nucleotide of the second strand, therebyforming a substantially duplexed region between the first and secondstrands; and the second strand is sufficiently complementary to a targetRNA along at least 19 ribonucleotides of the second strand length toreduce target gene expression when the double stranded nucleic acid isintroduced into a mammalian cell.

In an additional aspect, the invention provides an isolated doublestranded nucleic acid as shown in any one of FIG. 1-6, 8, 11, 14, or 15.

In one aspect, the invention provides a method for reducing expressionof a target gene in a cell, involving contacting a cell with an isolateddouble stranded nucleic acid as described herein in an amount effectiveto reduce expression of a target gene in a cell in comparison to areference dsRNA.

In another aspect, the invention provides a method for reducingexpression of a target gene in an animal, involving treating an animalwith an isolated double stranded nucleic acid as described herein in anamount effective to reduce expression of a target gene in a cell of theanimal in comparison to a reference dsRNA.

In one aspect, the invention provides a pharmaceutical composition forreducing expression of a target gene in a cell of a subject containingan isolated double stranded nucleic acid as described herein in anamount effective to reduce expression of a target gene in a cell incomparison to a reference dsRNA and a pharmaceutically acceptablecarrier.

In yet another aspect, the invention provides a method of synthesizing adouble stranded nucleic acid as described herein, involving chemicallyor enzymatically synthesizing the double stranded nucleic acid.

In still another aspect, the invention provides a kit containing thedouble stranded nucleic acid described herein and instructions for itsuse.

In various embodiments of any of the above aspects, the isolated doublestranded nucleic acid of claim 1, where at least one nucleotide of thefirst strand between and including the first strand positions 24 to the3′ terminal nucleotide residue of the first strand is adeoxyribonucleotide. In various embodiments of any of the above aspects,the isolated double stranded nucleic acid of claim 1, where at least 10consecutive nucleotides and at most 15 consecutive nucleotides, notincluding the unpaired 3′ terminal nucleotides of the second strand areunpaired with the first strand, thereby forming in the second strand a10-15 nucleotide single stranded 5′ overhang. In various embodiments ofany of the above aspects, the first strand is up to 30 nucleotides inlength, and the nucleotides of the first strand 3′ to position 23 of thefirst strand includes two, three, four, five, and six deoxynucleotideresidues from position 24 to the 3′ terminal nucleotide residue of thefirst strand that base pair with a nucleotide of the second strand.

In various embodiments of any of the above aspects, the 5′ singlestranded overhang of the second strand is 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides inlength. In various embodiments of any of the above aspects, thenucleotides of the second strand 5′ overhang include a phosphatebackbone modification. In various embodiments of any of the aboveaspects, the phosphate backbone modification is a phosphonate, aphosphorothioate, a phosphotriester, and a methylphosphonate, a lockednucleic acid, a morpholino, or a bicyclic furanose analog.

In various embodiments of any of the above aspects, the second strandstarting from the 5′ terminal nucleotide residue of the second strand(position 1^(B)), includes a phosphorothioate backbone modificationbetween the nucleotides from position 2¹³ to the 5′ residue of thesecond strand that corresponds to the 3′ terminal residue of the firststrand. In various embodiments of any of the above aspects, the secondstrand 5′ overhang includes a ribonucleotide or deoxyribonucleotide. Invarious embodiments of any of the above aspects, all nucleotides of thesecond strand 5′ overhang are deoxyribonucleotides. In variousembodiments of any of the above aspects, all nucleotides of the secondstrand 5′ overhang are ribonucleotides. In various embodiments of any ofthe above aspects, the nucleotides of the second strand 5′ overhanginclude a modified nucleotide. In various embodiments of any of theabove aspects, the modified nucleotide residue is 2′-O-methyl,2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge,4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino or 2′-O—(N-methlycarbamate). Invarious embodiments of any of the above aspects, the modified nucleotideof the second strand 5′ overhang is a 2′-O-methyl ribonucleotide. Invarious embodiments of any of the above aspects, the 5′ terminalnucleotide residue of the second strand is a 2′-O-methyl ribonucleotide.

In various embodiments of any of the above aspects, the isolated doublestranded nucleic acid, further includes a third oligonucleotide strandhaving a 5′ terminus and a 3′ terminus, where the third strand is 10-30nucleotide residues in length; where at least 10 consecutive nucleotidesand at most 30 consecutive nucleotides of the third strand are pairedwith the 5′ terminus of the second strand. In various embodiments of anyof the above aspects, the third strand includes a ribonucleotide ordeoxyribonucleotide. In various embodiments of any of the above aspects,all nucleotides of the second strand 5′ overhang are ribonucleotides. Invarious embodiments of any of the above aspects, the nucleotides of thethird strand include a modified nucleotide. In various embodiments ofany of the above aspects, the modified nucleotide residue is2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge,4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino and 2′-O—(N-methlycarbamate). Invarious embodiments of any of the above aspects, the modified nucleotideof the third strand is a 2′-O-methyl ribonucleotide. In variousembodiments of any of the above aspects, all nucleotides of the thirdstrand are modified nucleotides or 2′-O-methyl ribonucleotides. Invarious embodiments of any of the above aspects, the third strandincludes a phosphate backbone modification. In various embodiments ofany of the above aspects, the phosphate backbone modification is aphosphonate, a phosphorothioate, a phosphotriester, and amethylphosphonate, a locked nucleic acid, a morpholino, or a bicyclicfuranose analog. In various embodiments of any of the above aspects, thethird strand starting from the 5′ terminal nucleotide residue of thethird strand (position 1^(C)), includes a phosphorothioate backbonemodification between the nucleotides at positions 1^(C) and 2^(C).

In various embodiments of any of the above aspects, at least 10consecutive nucleotides and at most 15 consecutive nucleotides,including the 3′ terminal nucleotide of the first strand are unpairedwith the 5′ terminus of the second strand, thereby forming a 10-15nucleotide single stranded 3′ overhang. In various embodiments of any ofthe above aspects, the first strand is up to 66 nucleotides in length,and the nucleotides of the first strand 3′ to position 23 of the firststrand includes deoxyribonucleotides two, three, four, five, or sixdeoxynucleotide residues from positions 24 to the 3′ terminal nucleotideresidue of the first strand. In various embodiments of any of the aboveaspects, the deoxyribonucleotides are consecutive deoxyribonucleotides.In various embodiments of any of the above aspects, two or moreconsecutive nucleotide residues of positions 24 to 30 of the firststrand are deoxyribonucleotides that base pair with nucleotides of thesecond strand. In various embodiments of any of the above aspects, thefirst strand is up to 66 nucleotides in length and includes a pair ofdeoxyribonucleotides at positions 24 and 25, positions 25 and 26,positions 26 and 27, positions 27 and 28, positions 28 and 29, orpositions 29 and 30, where the first pair of deoxyribonucleotides isbase paired with a corresponding pair of nucleotides of the secondstrand.

In various embodiments of any of the above aspects, the isolated doublestranded nucleic acid of claim 67, where the first strand 3′ overhang is10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29 or 30 nucleotides in length. In various embodiments of any of theabove aspects, the first strand 3′ overhang includes a ribonucleotide ordeoxyribonucleotide. In various embodiments of any of the above aspects,all nucleotides of the first strand 3′ overhang aredeoxyribonucleotides. In various embodiments of any of the aboveaspects, the nucleotides of the first strand 3′ overhang include aphosphate backbone modification. In various embodiments of any of theabove aspects, the phosphate backbone modification is a phosphonate, aphosphorothioate, a phosphotriester, a methylphosphonate, a lockednucleic acid, a morpholino or a bicyclic furanose analog.

In various embodiments of any of the above aspects, the first strandstarting from the 3′ terminal nucleotide residue of the first strand(position 1^(D)), includes a methylphosphonate backbone modificationbetween the nucleotides from position 1^(D) to 5′ residue of the firststrand that is consecutive to the first strand 3′ overhang. In variousembodiments of any of the above aspects, the first strand starting fromthe 3′ terminal nucleotide residue of the first strand (position 1^(D)),includes a methylphosphonate backbone modification between thenucleotides from position 2^(D) to 5′ residue of the first strand thatis consecutive to the first strand 3′ overhang. In various embodimentsof any of the above aspects, the 3′ terminal nucleotide of the firststrand is a ribonucleotide.

In various embodiments of any of the above aspects, the nucleotides ofthe first strand 3′ overhang include a modified nucleotide. In variousembodiments of any of the above aspects, the modified nucleotide residueis 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge,4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino or 2′-O—(N-methlycarbamate). Invarious embodiments of any of the above aspects, the modified nucleotideof the first strand 3′ overhang is a 2′-O-methyl ribonucleotide.

In various embodiments of any of the above aspects, the first strand orthe second strand at least 8 ribonucleotides are contiguous. In variousembodiments of any of the above aspects, the first strand includes atleast 9, 10, 11, 12 and up to 25 ribonucleotides. In various embodimentsof any of the above aspects, the ribonucleotides are contiguous. Invarious embodiments of any of the above aspects, at least one nucleotideof the second strand between and including second strand nucleotidescorresponding to and thus base paired with first strand positions 24 tothe 3′ terminal nucleotide residue of the first strand is aribonucleotide.

In various embodiments of any of the above aspects, the substantiallyduplexed region between the first and second strands has a fullyduplexed region having no unpaired bases between the 5′ terminal and 3′terminal nucleotides of first strand that are paired with correspondingnucleotides of the second strand. In various embodiments of any of theabove aspects, the substantially duplexed region has, between the 5′terminal and 3′ terminal nucleotides of first strand that are pairedwith corresponding nucleotides of the second strand; 1 unpaired basepair; 2 unpaired base pairs, 3 unpaired base pairs, 4 unpaired basepairs, and 5 unpaired base pairs. In various embodiments of any of theabove aspects, the unpaired base pairs are consecutive ornon-consecutive.

In various embodiments of any of the above aspects, thedeoxyribonucleotides of the first strand that base pair with anucleotide of the second strand are consecutive deoxyribonucleotides. Invarious embodiments of any of the above aspects, at least one nucleotideof the first strand between and including the first strand positions 24to the 3′ terminal nucleotide residue of the first strand is adeoxyribonucleotide that base pairs with the second strand. In variousembodiments of any of the above aspects, two or more consecutivenucleotide residues of positions 24 to 30 of the first strand aredeoxyribonucleotides that base pair with nucleotides of the secondstrand. In various embodiments of any of the above aspects, the firststrand is up to 30 nucleotides in length and includes a pair ofdeoxyribonucleotides at positions 24 and 25, positions 25 and 26,positions 26 and 27, positions 27 and 28, positions 28 and 29, orpositions 29 and 30, where the first strand pair of deoxyribonucleotidesis base paired with a corresponding pair of nucleotides of the secondstrand.

In various embodiments of any of the above aspects, the 8 or moreribonucleotides of positions 1 to 28 of the first strand are consecutiveribonucleotides. In various embodiments of any of the above aspects,each nucleotide residue of positions 1 to 28 of the firstoligonucleotide strand is a ribonucleotide that base pairs with anucleotide of the second strand.

In various embodiments of any of the above aspects, the 3′ singlestranded overhang of the second strand is a length 1 to 4 nucleotides, 1to 3 nucleotides, 1 to 2 nucleotides, or 2 nucleotides in length. Invarious embodiments of any of the above aspects, the nucleotides of thesecond strand 3′ overhang includes a modified nucleotide. In variousembodiments of any of the above aspects, the modified nucleotide residueis a 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge,4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino or 2′-0-(N-methlycarbamate). Invarious embodiments of any of the above aspects, the modified nucleotideof the second strand 3′ overhang is a 2′-O-methyl ribonucleotide.

In various embodiments of any of the above aspects, all nucleotides ofthe second strand 3′ overhang are modified nucleotides. In variousembodiments of any of the above aspects, the second strand 3′ overhangis two nucleotides in length and where the modified nucleotide of thesecond strand 3′ overhang is a 2′-O-methyl modified ribonucleotide. Invarious embodiments of any of the above aspects, one or both of thefirst and second strands has a 5′ phosphate.

In various embodiments of any of the above aspects, the second strand,starting from the nucleotide residue of the second strand thatcorresponds to the 5′ terminal nucleotide residue of the firstoligonucleotide strand (position 1^(A)), includes unmodified nucleotideresidues at all positions from position 16^(A) to the 5′ residue of thesecond strand that corresponds to the 3′ terminal residue of the firststrand. In various embodiments of any of the above aspects, startingfrom the first nucleotide (position 1^(A)) at the 3′ terminus of thesecond strand, positions 1^(A), 2^(A), and 3^(A) from the 3′ terminus ofthe second strand are modified nucleotides. In various embodiments ofany of the above aspects, the second oligonucleotide strand, startingfrom the nucleotide residue of the second strand that corresponds to the5′ terminal nucleotide residue of the first oligonucleotide strand(position 1^(A)), includes alternating modified and unmodifiednucleotide residues from position 1^(A) to position 15^(A).

In various embodiments of any of the above aspects, a nucleotide of thesecond or first oligonucleotide strand is substituted with a modifiednucleotide that directs the orientation of Dicer cleavage. In variousembodiments of any of the above aspects, the first strand has anucleotide sequence that is at least 80%, 90%, 95% or 100% complementaryto the second strand nucleotide sequence. In various embodiments of anyof the above aspects, the double stranded nucleic acid is cleavedendogenously in a mammalian cell by Dicer. In various embodiments of anyof the above aspects, the double stranded nucleic acid is cleavedendogenously in a mammalian cell to produce a double-stranded nucleicacid of 19-23 nucleotides in length that reduces target gene expression.In various embodiments of any of the above aspects, the double strandednucleic acid reduces target gene expression in a mammalian cell in vitroby an amount (expressed by %) at least 10%, at least 50% or at least80-90%. In various embodiments of any of the above aspects, the doublestranded nucleic acid, when introduced into a mammalian cell, reducestarget gene expression in comparison to a reference dsRNA that does notpossess a deoxyribonucleotide-deoxyribonucleotide base pair. In variousembodiments of any of the above aspects, where the double strandednucleic acid, when introduced into a mammalian cell, reduces target geneexpression by at least 70% when transfected into the cell at aconcentration of 1 nM or less, 200 pM or less, 100 pM or less, 50 pM orless, 20 pM or less or 10 pM or less. In various embodiments of any ofthe above aspects, at least 50% of the ribonucleotide residues of thedouble stranded nucleic acid are unmodified ribonucleotides. In variousembodiments of any of the above aspects, at least 50% of theribonucleotide residues of the second strand are unmodifiedribonucleotides. In various embodiments of any of the above aspects, thetarget RNA is KRAS.

In various embodiments of any of the above aspects, double strandednucleic acid possesses enhanced pharmacokinetics when compared to anappropriate control DsiRNA. In various embodiments of any of the aboveaspects, the double stranded nucleic acid possesses enhancedpharmacodynamics when compared to an appropriate control DsiRNA. Invarious embodiments of any of the above aspects, the double strandednucleic acid possesses reduced toxicity when compared to an appropriatecontrol DsiRNA. In various embodiments of any of the above aspects, thedouble stranded nucleic acid possesses enhanced intracellular uptakewhen compared to an appropriate control DsiRNA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the structure and predicted Dicer-mediatedprocessing of exemplary single strand extended Dicer substrates. In FIG.1A, Panel A depicts a DsiRNA without a single stranded extension. PanelB depicts a “guide strand extended” DsiRNA agent, which has a guidestrand 5′ overhang 1-30 nucleotides in length (15 nucleotides as shown).Panel C depicts an exemplary “guide strand extended” DsiRNA agent, whichhas a guide strand 5′ overhang 1-30 nucleotides in length (15nucleotides as shown), with a short oligo complementary to thesingle-stranded extended region (“discontinuous complement”;discontinuous 3′ passenger complement as shown). Panel D depicts anexemplary “passenger strand extended” DsiRNA agent, which has apassenger strand 3′ overhang 1-30 nucleotides in length (15 nucleotidesas shown). Panel E depicts a “passenger strand extended” DsiRNA agent,which has a passenger strand 5′ overhang 1-30 nucleotides in length (15nucleotides as shown). In each pair of oligonucleotide strands forming aDsiRNA, the upper strand is the passenger strand and the lower strand isthe guide strand. White=nucleotide (e.g., a ribonucleotide,deoxyribonucleotide, modified ribonucleotide). FIG. 1B shows nucleotidemodifications and patterns of modifications of exemplary single strandextended Dicer substrates. Panel A depicts a DsiRNA without a singlestranded extension. Panel B depicts a “guide strand extended” DsiRNAagent, which has a guide strand 5′ overhang 1-30 nucleotides in length(15 nucleotides as shown). Panel C depicts an exemplary “guide strandextended” DsiRNA agent, which has a guide strand 5′ overhang 1-30nucleotides in length (15 nucleotides as shown), with a short oligocomplementary to the single-stranded extended region (“discontinuouscomplement”; discontinuous 3′ passenger complement as shown). Panel Ddepicts an exemplary “passenger strand extended” DsiRNA agent, which hasa passenger strand 3′ overhang 1-30 nucleotides in length (15nucleotides as shown). In each pair of oligonucleotide strands forming aDsiRNA, the upper strand is the passenger strand and the lower strand isthe guide strand. Blue=ribonucleotide or modified ribonucleotide (e.g.,2′-O-methyl ribonucleotide); Gray=deoxyribonucleotide or ribonucleotide;White=ribonucleotide; Dark Yellow=deoxyribonucleotide, ribonucleotide,or modified nucleotide (e.g., 2′-O-methyl ribonucleotide,phosophorothioate deoxyribonucleotide; methylphosphonatedeoxyribonucleotide). Small arrow=Dicer cleavage site; largearrow=discontinuity. ^(A)=position starting from the nucleotide residueof guide strand that is complementary to the 5′ terminal nucleotideresidue of passenger strand (position 1^(A)); ^(B)=position startingfrom the 5′ terminal nucleotide residue of guide strand (position1^(B)); ^(C)=position starting from the 5′ terminal nucleotide of theshort oligo complementary to single-stranded extended region (position1^(C)); ^(D)=position starting from the 3′ terminal nucleotide residueof passenger strand (position 1^(D)); ^(E)=position starting from the 3′terminal nucleotide residue of passenger strand (position 1E);^(F)=position starting from the 5′ terminal nucleotide consecutive tothe first strand 5′ single stranded overhang (position 1^(E)). Smallarrows indicate predicted Dicer cleavage sites; a large arrow indicatesa discontinuity.

FIG. 2 shows the structure and predicted Dicer-mediated processing ofexemplary “guide strand extended” DsiRNA agents, which have a guidestrand 5′ overhang 1-30 nucleotides in length (10-15 nucleotides asshown). Blue=2′-O-methyl ribonucleotide; Gray=deoxyribonucleotide;White=ribonucleotide; Dark Yellow=phosophorothioate deoxyribonucleotide;Green=phosphorothioate 2′-O-methyl ribonucleotide; Pink=phosphorothioateribonucleotide; Light Yellow=methylphosphonate deoxyribonucleotide.^(A)=position starting from the nucleotide residue of said second strandthat is complementary to the 5′ terminal nucleotide residue of passengerstrand (position 1^(A)); ^(B)=position starting from the 5′ terminalnucleotide residue of guide strand (position 1^(B)). Arrows indicatepredicted Dicer cleavage sites.

FIG. 3 shows the structure and predicted Dicer-mediated processing ofexemplary “passenger strand extended” DsiRNA agents, which have apassenger strand 3′ overhang 1-30 nucleotides in length (10-15nucleotides, as shown). Blue=2′-O-methyl ribonucleotide;Gray=deoxyribonucleotide; White=ribonucleotide; DarkYellow=phosophorothioate deoxyribonucleotide; Green=phosphorothioate2′-O-methyl ribonucleotide; Pink=phosphorothioate ribonucleotide; LightYellow=methylphosphonate deoxyribonucleotide. ^(A)=position startingfrom the nucleotide residue of said second strand that is complementaryto the 5′ terminal nucleotide residue of passenger strand (position1^(A)); ^(D)=position starting from the 3′ terminal nucleotide residueof passenger strand. Arrows indicate predicted Dicer cleavage sites.

FIG. 4 shows the structure and predicted Dicer-mediated processing ofexemplary “guide strand extended” DsiRNA agents, which have a guidestrand 5′ overhang 1-30 nucleotides in length. Single stranded guideextended DsiRNA agents having a passenger strand with the modificationpattern depicted by DP1301P and a guide strand with a modificationpattern depicted by DP1337G; DP1339G; DP1371G; and DP 1338G weregenerated. Additionally, the single stranded extended DsiRNA agentshaving a passenger strand with the modification pattern depicted inDP1301P, a guide strand with a modification pattern depicted by DP1337G;DP1339G; DP1371G; and DP1338G, and a “discontinuous 3′ passengercomplement” strand with a modification pattern depicted by DP1372P andDP1373P were generated. DsiRNA agents having a guide strand with themodification depicted DP1370G were used as a reference. Blue=2′-O-methylribonucleotide; Gray=deoxyribonucleotide; White=ribonucleotide; DarkYellow=phosophorothioate deoxyribonucleotide; Green=phosphorothioate2′-O-methyl ribonucleotide; Pink=phosphorothioate ribonucleotide; LightYellow=methylphosphonate deoxyribonucleotide. Arrows indicate predictedDicer cleavage sites.

FIG. 5 shows the structure and predicted Dicer-mediated processing ofexemplary “passenger strand extended” DsiRNA agents, which have apassenger strand 3′ overhang 1-30 nucleotides in length. Single strandedpassenger extended DsiRNA agents having a guide strand with themodification pattern depicted by DP1XXXG and a passenger strand with amodification pattern depicted by DP1YYXP; DP1YxxP; and DP1YxxP weregenerated. DsiRNA agents having a passenger strand with the modificationdepicted DP1301P were used as a reference. Blue=2′-O-methylribonucleotide; Gray=deoxyribonucleotide; White=ribonucleotide; DarkYellow=phosophorothioate deoxyribonucleotide; Green=phosphorothioate2′-O-methyl ribonucleotide; Pink=phosphorothioate ribonucleotide; LightYellow=methylphosphonate deoxyribonucleotide. Arrows indicate predictedDicer cleavage sites.

FIGS. 6A and B shows the sequence, structure, and predictedDicer-mediated processing of exemplary “guide strand extended” DsiRNAagents targeting KRAS-249M, which have a guide strand 5′ overhang 1-15nucleotides in length. FIG. 6B shows single stranded guide extendedDsiRNA agents having a passenger strand depicted by DP1301P and a guidestrand depicted by DP1337G; DP1338G; DP1340G; DP1341G; and DP1342G weregenerated and tested. DsiRNA agents having a passenger strand depictedby DP1301P and a guide strand depicted by DP1336G were used as areference (FIG. 6A). Descriptions of the modification patterns of thediscontinuous complements are labeled to the right. RNA=ribonucleotide;PS=phosphorothioate; DNA=deoxyribonucleotide; 2′OMe=2′-O-methyl;Underline=2′-O-methyl ribonucleotide; Bold=guide strand 5′ overhang;lower=deoxyribonucleotide; UPPER=ribonucleotide. Arrows indicatepredicted Dicer cleavage sites.

FIG. 7 is a histogram showing the normalized fold expression ofKRAS-249M using DsiRNA agents having the passenger strands and guidestrands depicted in FIG. 5. Hela cells were treated with 0.1 nM of theDsiRNA agents in RNAiMAX, 24 hrs.

FIGS. 8A and B shows the sequence, structure, and predictedDicer-mediated processing of exemplary “guide strand extended” DsiRNAagents targeting HPRT1, which have a guide strand 5′ overhang 1-15nucleotides in length. FIG. 8B presents single stranded guide extendedDsiRNA agents having a passenger strand depicted by DP1001P and a guidestrand depicted by DP1350G; DP1351G; DP1352G; DP1353G; DP1354G; andDP1355G were generated and tested. DsiRNA agents having a passengerstrand depicted by DP1001P and a guide strand depicted by DP1002G wereused as a reference (FIG. 8A). Descriptions of the modification patternsof the discontinuous complements are labeled to the right.RNA=ribonucleotide; PS=phosphorothioate; DNA=deoxyribonucleotide;2′OMe=2′-O-methyl; Underline=2′-O-methyl ribonucleotide; Bold=guidestrand 5′ overhang; lower=deoxyribonucleotide; UPPER=ribonucleotide.Arrows indicate predicted Dicer cleavage sites.

FIG. 9 is a histogram showing the normalized fold expression of HPRT1using DsiRNA agents having the passenger strands and guide strandsdepicted in FIG. 7. Hela cells were treated with 0.1 nM of the DsiRNAagents in RNAiMAX, 24 hrs.

FIG. 10 is an image of a gel showing a Dicer activity on single strandedguide extended DsiRNA agents (passenger+guide strands) targetingKRAS-249M or HPRT1. Treatment: 2 h @ 37 C Turbo Dicer (1 U/reaction).Gel: 18% Tris 90′ @ 10 W. Loading: (1 μl 50 μM+50 μl Buffer and load 10μl) or (5 μl reaction+20 μl Buffer and load 10 μl).

FIG. 11 shows the sequence and structure of exemplary short oligos thatcomplement guide strand extensions (“discontinuous complements”), whichare 1-16 nucleotides in length, base paired to 5′ guide strandextensions. Single stranded guide extended DsiRNA agents having adiscontinuous complement depicted by DP1365P; DP1366P; DP1367P; DP1368P;and DP1369P were generated and tested. Descriptions of the modificationpatterns of the discontinuous complements are labeled to the right.RNA=ribonucleotide; PS=phosphorothioate; DNA=deoxyribonucleotide;2′OMe=2′-O-methyl; Underline=2′-O-methyl ribonucleotide; Bold=guidestrand 5′ overhang; lower=deoxyribonucleotide; UPPER=ribonucleotide.Arrows indicate predicted Dicer cleavage sites.

FIG. 12 is a histogram showing the normalized fold expression ofKRAS-249M using DsiRNA agents having the passenger strands and guidestrands depicted in FIG. 7 and the discontinuous complements depicted inFIG. 10. The discontinuous complements used is labeled above each set ofthe three bars corresponding to DsiRNA agents having a 5′ guide singlestranded extension (l.-r. DNA, RNA, 2′OMe RNA). Hela cells were treatedwith 0.1 nM of the DsiRNA agents in RNAiMAX, 24 hrs.

FIG. 13 is a histogram showing the normalized fold expression of HPRT1using DsiRNA agents having the passenger strands and guide strandsdepicted in FIG. 7 and the discontinuous complements depicted in FIG.10. The discontinuous complement used is labeled above each set of thethree bars corresponding to DsiRNA agents having a 5′ guide singlestranded extension (1.-r. DNA, RNA, 2′OMe RNA). Hela cells were treatedwith 0.1 nM of the DsiRNA agents in RNAiMAX, 24 hrs.

FIG. 14 show the structure and predicted Dicer-mediated processing ofexemplary single strand extended Dicer substrates in an in vivoexperiment (Experimental conditions: Treatment: 10 mg/kg one injection;Target: KRAS; Transfection: invivoFectamine; Tissue: Liver). Panel Adepicts a modification pattern used in a negative control DsiRNA withouta single stranded extension. Panel B depicts a modification pattern usedin a positive control DsiRNA without a single stranded extension. PanelC depicts a modification pattern used in a test DsiRNA without a singlestranded extension. Panel D depicts a modification pattern used in atest “guide strand extended” DsiRNA agent, which has a guide strand 5′overhang 1-30 nucleotides in length (10 nucleotides as shown). Panel Edepicts a modification pattern used in a test “guide strand extended”DsiRNA agent, which has a guide strand 5′ overhang 1-30 nucleotides inlength (10 nucleotides as shown). In each pair of oligonucleotidestrands forming a DsiRNA, the upper strand is the passenger strand andthe lower strand is the guide strand. Blue=ribonucleotide or modifiedribonucleotide (e.g., 2′-O-methyl ribonucleotide);Gray=deoxyribonucleotide or ribonucleotide; White=ribonucleotide; DarkYellow=deoxyribonucleotide, ribonucleotide, or modified nucleotide(e.g., 2′-O-methyl ribonucleotide, phosophorothioatedeoxyribonucleotide; methylphosphonate deoxyribonucleotide). Smallarrow=Dicer cleavage site; large arrow=discontinuity. ^(A)=positionstarting from the nucleotide residue of said second strand that iscomplementary to the 5′ terminal nucleotide residue of passenger strand(position 1^(A)); ^(B)=position starting from the 5′ terminal nucleotideresidue of guide strand. Small arrows indicate predicted Dicer cleavagesites; a large arrow indicates a discontinuity.

FIGS. 15A and B shows the sequence, structure, and predictedDicer-mediated processing of exemplary “guide strand extended” DsiRNAagents targeting KRAS-249M and HPRT1, which have a guide strand 5′overhang 1-15 nucleotides in length. The sequence and structure ofexemplary short oligos that complement guide strand extensions(“discontinuous complements”) are shown base paired to 5′ guide strandextension sequences (FIGS. 15 A and B). Single stranded guide extendedDsiRNA agents having a passenger strand with the modification patterndepicted by DP1301P and a guide strand with a modification patterndepicted by DP1337G, DP1338G, DP1339G, DP1371G, and DP1352G weregenerated. Additionally, the single stranded extended DsiRNA agentshaving a passenger strand with the modification pattern depicted inDP1301P, a guide strand with a modification pattern depicted by DP1337G,DP1338G, DP1339G, DP1371G, and DP1352G; and an “discontinuouscomplement” strand with a modification pattern depicted by DP1372P andDP1373P were generated. DsiRNA agents having a passenger strand with themodification depicted by DP1301P were used as a reference and a guidestrand with the modification depicted by DP1370G were used as areference. Dosage of passenger strands, guide strands, and discontinuouscomplements are labeled to the right. Descriptions of the modificationpatterns of the discontinuous complements are also labeled on the right.RNA=ribonucleotide; PS=phosphorothioate; DNA=deoxyribonucleotide;2′OMe=2′-O-methyl; Underline=2′-O-methyl ribonucleotide; Bold=guidestrand 5′ overhang; lower=deoxyribonucleotide; UPPER=ribonucleotide.Arrows indicate predicted Dicer cleavage sites.

FIG. 16 is a histogram showing the normalized fold expression of mKRASin liver of individual animals treated with DsiRNA agents having thepassenger strands and guide strands depicted in FIGS. 14 and/or 15Animals were treated with a 10 mg/kg injection of the DsiRNA agents ininvivoFectamine and liver samples were analyzed.

FIG. 17 is a histogram showing the normalized fold expression of mKRASin liver of animals treated with DsiRNA agents having the passengerstrands and guide strands depicted in FIGS. 14 and/or 15 Animals weretreated with a 10 mg/kg injection of the DsiRNA agents in invivoFectamine and liver samples were analyzed.

FIG. 18 are graphs showing the normalized fold expression of mKRAS inliver of animals treated with DsiRNA agents having the passenger strandsand guide strands depicted in FIGS. 14 and/or 15 Animals were treatedwith a 10 mg/kg injection of the DsiRNA agents in in vivoFectamine andliver samples were analyzed.

FIG. 19 is a histogram showing the normalized fold expression of mKRASin spleen of individual animals treated with DsiRNA agents having thepassenger strands and guide strands depicted in FIGS. 14 and/or 15Animals were treated with a 10 mg/kg injection of the DsiRNA agents inin vivoFectamine and spleen samples were analyzed.

FIG. 20 is a histogram showing the normalized fold expression of mKRASin spleen of animals treated with DsiRNA agents having the passengerstrands and guide strands depicted in FIGS. 14 and/or 15 Animals weretreated with a 10 mg/kg injection of the DsiRNA agents ininvivoFectamine and spleen samples were analyzed.

FIG. 21 are graphs showing the normalized fold expression of mKRAS inspleen of animals treated with DsiRNA agents having the passengerstrands and guide strands depicted in FIGS. 14 and/or 15 Animals weretreated with a 10 mg/kg injection of the DsiRNA agents in invivoFectamine and spleen samples were analyzed.

FIG. 22 is a histogram showing the normalized fold expression of mKRASin kidney of individual animals treated with DsiRNA agents having thepassenger strands and guide strands depicted in FIGS. 14 and/or 15Animals were treated with a 10 mg/kg injection of the DsiRNA agents inin vivoFectamine and kidney samples were analyzed.

FIG. 23 is a histogram showing the normalized fold expression of mKRASin kidney of animals treated with DsiRNA agents having the passengerstrands and guide strands depicted in FIGS. 14 and/or 15 Animals weretreated with a 10 mg/kg injection of the DsiRNA agents ininvivoFectamine and kidney samples were analyzed.

FIG. 24 are graphs showing the normalized fold expression of mKRAS inkidney of animals treated with DsiRNA agents having the passengerstrands and guide strands depicted in FIGS. 14 and/or 15 Animals weretreated with a 10 mg/kg injection of the DsiRNA agents ininvivoFectamine and kidney samples were analyzed.

DETAILED DESCRIPTION

The invention provides compositions and methods for reducing expressionof a target gene in a cell, involving contacting a cell with an isolateddouble stranded nucleic acid in an amount effective to reduce expressionof a target gene in a cell. The dsNAs of the invention possess a singlestranded nucleotide region either at the 5′ terminus of the antisensestrand or at the 3′ terminus of the sense strand are effective RNAinterference agents (in most embodiments, the single stranded extensioncomprises at least one modified nucleotide and/or phosphate back bonemodification). Surprisingly, as demonstrated herein, single-strandedextended Dicer-substrate siRNAs (DsiRNAs) were effective RNA inhibitoryagents when compared to corresponding DsiRNAs.

The surprising discovery that single stranded extended DsiRNA agents donot exhibit decreases in efficacy allows for the generation of DsiRNAsthat remain effective while providing greater spacing for, e.g.,attachment of DsiRNAs to additional and/or distinct functional groups,inclusion/patterning of stabilizing modifications (e.g., PS-NA moieties)or other forms of modifications capable of adding further functionalityand/or enhancing, e.g., pharmacokinetics, pharmacodynamics orbiodistribution of such agents, as compared to dsRNA agents ofcorresponding length that do not contain such single strandedDNA-extended domains.

The advantage provided by the newfound ability to lengthen either the 5′guide strand, the 3′ passenger strand, or the 5′ passenger strand ofDsiRNA-containing dsNA duplexes while retaining activity of apost-Dicer-processed siRNA agent at levels greater than dsRNA duplexesof similar length is emphasized by the results presented herein. Theability to extend either the 5′ guide strand, the 3′ passenger strand,or 5′ passenger strand of DsiRNA agents without observing acorresponding reduction in RNA silencing activity can also allow forcertain functional groups to be attached to such agents that wouldotherwise not be possible, because of the ability of such functionalgroups to interfere with RNA silencing activity when present in tighterconfigurations.

Additionally, single stranded extended DsiRNA agents may include a thirdshort (1-16 nucleotides in length) oligonucleotide which base-pairs withthe single stranded region of a single extended DsiRNAs, e.g., whichbase-pairs to a guide 5′ single stranded extended region. The thirdoligo provides advantages to the use of single stranded extended DsiRNAagents: (a) to stabilize the single stranded extension (without beingbound to a particular theory, the single strand extended DsiRNA might berapidly degraded) and (b) to provide an independent entity to which atargeting molecule (or other active agent) could be attached, whichcould then be joined to the single-stranded extended DsiRNA viaannealing (versus direct attachment of the targeting molecule to thesingle stranded extended DsiRNA).

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

As used herein, the term “nucleic acid” refers to deoxyribonucleotides,ribonucleotides, or modified nucleotides, and polymers thereof insingle- or double-stranded form. The term encompasses nucleic acidscontaining known nucleotide analogs or modified backbone residues orlinkages, which are synthetic, naturally occurring, and non-naturallyoccurring, which have similar binding properties as the referencenucleic acid, and which, in certain cases, are metabolized in a mannersimilar to the reference nucleotides. Examples of such analogs include,without limitation, phosphorothioates, phosphoramidates,methylphosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs).

As used herein, “nucleotide” is used as recognized in the art to includethose with natural bases (standard), and modified bases well known inthe art. Such bases are generally located at the 1′ position of anucleotide sugar moiety. Nucleotides generally comprise a base, sugarand a phosphate group. The nucleotides can be unmodified or modified atthe sugar, phosphate and/or base moiety, (also referred tointerchangeably as nucleotide analogs, modified nucleotides, non-naturalnucleotides, non-standard nucleotides and other; see, e.g., Usman andMcSwiggen, supra; Eckstein, et al., International PCT Publication No. WO92/07065; Usman et al, International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach, et al, Nucleic Acids Res. 22:2183,1994. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, hypoxanthine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin, et al., Biochemistry 35:14090, 1996; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

As used herein, a “double-stranded nucleic acid” or “dsNA” is a moleculecomprising two oligonucleotide strands which form a duplex. A dsNA maycontain ribonucleotides, deoxyribonucleotides, modified nucleotides, andcombinations thereof. The double-stranded NAs of the instant inventionare substrates for proteins and protein complexes in the RNAinterference pathway, e.g., Dicer and RISC. An exemplary structure ofone form of dsNA of the invention is shown in FIG. 1A, Panel B, and suchstructures characteristically comprise an RNA duplex in a region that iscapable of functioning as a Dicer substrate siRNA (DsiRNA) and a singlestranded region, which is located at a position 5′ of the projectedDicer cleavage site of the second strand of the DsiRNA/DNA agent. Anexemplary structure of another form of dsNA of the invention is shown inFIG. 1A, Panel C, and such structures characteristically comprise an RNAduplex in a region that is capable of functioning as a Dicer substratesiRNA (DsiRNA) and a single stranded region, which is located at aposition 3′ of the projected Dicer cleavage site of the first strand ofthe DsiRNA/DNA agent. In further embodiments, the instant inventionprovides a structure that characteristically comprises an RNA duplexthat is capable of functioning as a Dicer substrate siRNA (DsiRNA) and asingle stranded region comprising at least one modified nucleotideand/or phosphate backbone modification, which is located at a position3′ of the projected Dicer cleavage site of the second strand of theDsiRNA/DNA agent. In alternative embodiments, the instant inventionprovides a structure that characteristically comprises an RNA duplexthat is capable of functioning as a Dicer substrate siRNA (DsiRNA) and asingle stranded region comprising at least one modified nucleotideand/or phosphate backbone modification, which is located at a position5′ of the projected Dicer cleavage site of the first strand of theDsiRNA/DNA agent

In certain embodiments, the DsiRNAs of the invention can possessdeoxyribonucleotide residues at sites immediately adjacent to theprojected Dicer enzyme cleavage site(s). For example, in the all theDsiRNAs shown in FIG. 2 and in the sixth, seventh, eighth, ninth, tenth,eleventh, and twelfth DsiRNAs shown in FIG. 3, deoxyribonucleotides canbe found (starting at the 5′ terminal residue of the first strand asposition 1) at position 24 and sites 3′ of position 24 (e.g., 24, 25,26, 27, 28, 29, 30, etc.). Deoxyribonucleotides may also be placed onthe second strand commencing at the nucleotide that is complementary toposition 20 of the first strand, and also at positions on the secondstrand that are located in the 5′ direction of this nucleotide. Thus,certain effective DsiRNAs of the invention possess only 19 duplexedribonucleotides prior to commencement of introduction ofdeoxyribonucleotides within the first strand, second strand, and/or bothstrands of such DsiRNAs.

As used herein, “duplex” refers to a double helical structure formed bythe interaction of two single stranded nucleic acids. According to thepresent invention, a duplex may contain first and second strands whichare sense and antisense, or which are target and antisense. A duplex istypically formed by the pairwise hydrogen bonding of bases, i.e., “basepairing”, between two single stranded nucleic acids which are orientedantiparallel with respect to each other. As used herein, the term“duplex” refers to the regions of the first and second strands whichalign such that if the aligned bases of the strands are complementary,they may Watson-Crick base pair. The term “duplex” does not include oneor more single stranded nucleotides which includes a 5′ or 3′ terminalsingle stranded nucleotide. The term “duplex” includes a region ofaligned first and second strands which may be fully (100%) base pairedand a region of aligned first and second strands which contains 1, 2, 3,4, or 5 unpaired bases, as long as the first strand 5′ terminalnucleotide and the first strand 3′ terminal nucleotide are Watson-Crickbase paired with a corresponding nucleotide of the second strand. Asused herein, “fully duplexed” refers to all nucleotides in between thepaired 5′ and 3′ terminal nucleotides are base-paired. As used herein,“substantially duplexed” refers to a duplex between the strands suchthat there is 1, 2, 3, 4, 5 unpaired base pair(s) (consecutive ornon-consecutive) between the between the 5′ terminal and 3′ terminalnucleotides of the first strand.

Pairing in duplexes generally occurs by Watson-Crick base pairing, e.g.,guanine (G) forms a base pair with cytosine (C) in DNA and RNA (thus,the cognate nucleotide of a guanine deoxyribonucleotide is a cytosinedeoxyribonucleotide, and vice versa), adenine (A) forms a base pair withthymine (T) in DNA, and adenine (A) forms a base pair with uracil (U) inRNA. Conditions under which base pairs can form include physiological orbiologically relevant conditions (e.g., intracellular: pH 7.2, 140 mMpotassium ion; extracellular pH 7.4, 145 mM sodium ion). Furthermore,duplexes are stabilized by stacking interactions between adjacentnucleotides. As used herein, a duplex may be established or maintainedby base pairing or by stacking interactions. A duplex is formed by twocomplementary nucleic acid strands, which may be substantiallycomplementary or fully complementary (see below).

As used herein, “corresponds to” or “corresponding to” refers to firstand second strand bases that are aligned in a duplex such that thenucleotide residue of the second strand aligns with the residue of thefirst strand, when first strand position 1 is base paired with anucleotide of said second strand such that said second strand comprisesa 3′ single stranded overhang of 1-6 nucleotides in length. “Correspondsto” does not require pairing via formation of a Watson-Crick base pair,but rather includes both aligned and unpaired first strand/second strandnucleotides as well as aligned and base paired first strand/secondstrand nucleotides.

By “complementary” or “complementarity” is meant that a nucleic acid canform hydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick or Hoogsteen base pairing. In reference to thenucleic acid molecules of the present disclosure, the binding freeenergy for a nucleic acid molecule with its complementary sequence issufficient to allow the relevant function of the nucleic acid toproceed, e.g., RNAi activity. Determination of binding free energies fornucleic acid molecules is well known in the art (see, e.g., Turner, etal., CSH Symp. Quant. Biol. LII, pp. 123-133, 1987; Frier, et al., Proc.Nat. Acad. Sci. USA 83:9373-9377, 1986; Turner, et al., J. Am. Chem.Soc. 109:3783-3785, 1987). A percent complementarity indicates thepercentage of contiguous residues in a nucleic acid molecule that canform hydrogen bonds (e.g., Watson-Crick base pairing) with a secondnucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of atotal of 10 nucleotides in the first oligonucleotide being based pairedto a second nucleic acid sequence having 10 nucleotides represents 50%,60%, 70%, 80%, 90%, and 100% complementary, respectively). To determinethat a percent complementarity is of at least a certain percentage, thepercentage of contiguous residues in a nucleic acid molecule that canform hydrogen bonds (e.g., Watson-Crick base pairing) with a secondnucleic acid sequence is calculated and rounded to the nearest wholenumber (e.g., 12, 13, 14, 15, 16, or 17 nucleotides out of a total of 23nucleotides in the first oligonucleotide being based paired to a secondnucleic acid sequence having 23 nucleotides represents 52%, 57%, 61%,65%, 70%, and 74%, respectively; and has at least 50%, 50%, 60%, 60%,70%, and 70% complementarity, respectively). As used herein,“substantially complementary” refers to complementarity between thestrands such that they are capable of hybridizing under biologicalconditions. Substantially complementary sequences have 60%, 70%, 80%,90%, 95%, or even 100% complementarity. Additionally, techniques todetermine if two strands are capable of hybridizing under biologicalconditions by examining their nucleotide sequences are well known in theart.

As used herein, the “3′ region” with respect to the antisense strandrefers to the consecutive nucleotides of the antisense strand that are3′ distal (on the antisense strand) to the nucleotide of the antisensestrand that aligns with corresponding positions 1-19, 1-20 or 1-21 ofthe sense strand. To avoid doubt, the “3′ region”, when referring to theantisense strand, is meant to encompass antisense nucleotides in aduplex formed between the antisense strand and its cognate target RNA 3′distal to (on the antisense strand which correspond to nucleotides onthe target RNA that are 5′ distal to) the projected Argonaute 2 (Ago2)cut site.

The first and second strands of the agents of the invention (sense andantisense oligonucleotides) are not required to be completelycomplementary in the duplexed region. In one embodiment, the RNAsequence of the antisense strand contains one or more mismatches (1, 2,3, 4 or 5, consecutive or nonconsecutive), i.e., mismatched with respectto the duplexed sense strand of the isolated double stranded nucleicacid according to the invention, contains one or more (1, 2, 3, 4 or 5,consecutive or nonconsecutive), modified nucleotides (base analog)s. Inan exemplary embodiment, such mismatches occur within the 3′ region, asdefined hereinabove, of RNA sequence of the antisense strand. In oneaspect, two, three, four or five mismatches or modified nucleotides withbase analogs are incorporated within the RNA sequence of the antisensestrand that is 3′ in the antisense strand of the projected Ago2 cleavagesite of the target RNA sequence when the target RNA sequence ishybridized.

The use of mismatches or decreased thermodynamic stability (specificallyat or near the 3′-terminal residues of sense/5′-terminal residues of theantisense region of siRNAs) has been proposed to facilitate or favorentry of the antisense strand into RISC (Schwarz et al., 2003; Khvorovaet al., 2003), presumably by affecting some rate-limiting unwindingsteps that occur with entry of the siRNA into RISC. Thus, terminal basecomposition has been included in design algorithms for selecting active21mer siRNA duplexes (Ui-Tei et al., 2004; Reynolds et al., 2004).

Inclusion of such mismatches within the DsiRNA agents of the instantinvention can allow such agents to exert inhibitory effects thatresemble those of naturally-occurring miRNAs, and optionally can bedirected against not only naturally-occurring miRNA target RNAs (e.g.,3′ UTR regions of target transcripts) but also against RNA sequences forwhich no naturally-occurring antagonistic miRNA is known to exist. Forexample, DsiRNAs of the invention possessing mismatched base pairs whichare designed to resemble and/or function as miRNAs can be synthesized totarget repetitive sequences within genes/transcripts that might not betargeted by naturally-occurring miRNAs (e.g., repeat sequences withinthe Notch protein can be targeted, where individual repeats within Notchcan differ from one another (e.g., be degenerate) at the nucleic acidlevel, but which can be effectively targeted via a miRNA mechanism thatallows for mismatch(es) yet also allows for a more promiscuousinhibitory effect than a corresponding, perfect match siRNA agent). Insuch embodiments, target RNA cleavage may or may not be necessary forthe mismatch-containing DsiRNA agent to exert an inhibitory effect.

In one embodiment, a double stranded nucleic acid molecule of theinvention comprises or functions as a microRNA (miRNA). By “microRNA” or“miRNA” is meant a small double stranded RNA that regulates theexpression of target messenger RNAs either by mRNA cleavage,translational repression/inhibition or heterochromatic silencing (seefor example Ambros, 2004, Nature, 431, 350-355; Bartel, 2004, Cell, 116,281-297; Cullen, 2004, Virus Research., 102, 3-9; He et al., 2004, Nat.Rev. Genet., 5, 522-531; and Ying et al., 2004, Gene, 342, 25-28). Inone embodiment, the microRNA of the invention, has partialcomplementarity (i.e., less than 100% complementarity) between the sensestrand (e.g., first strand) or sense region and the antisense strand(e.g., second strand) or antisense region of the miRNA molecule orbetween the antisense strand or antisense region of the miRNA and acorresponding target nucleic acid molecule (e.g., target mRNA). Forexample, partial complementarity can include various mismatches ornon-base paired nucleotides (e.g., 1, 2, 3, 4, 5 or more mismatches ornon-based paired nucleotides, such as nucleotide bulges) within thedouble stranded nucleic acid molecule structure, which can result inbulges, loops, or overhangs that result between the sense strand orsense region and the antisense strand or antisense region of the miRNAor between the antisense strand or antisense region of the miRNA and acorresponding target nucleic acid molecule.

Single-stranded nucleic acids that base pair over a number of bases aresaid to “hybridize.” Hybridization is typically determined underphysiological or biologically relevant conditions (e.g., intracellular:pH 7.2, 140 mM potassium ion; extracellular pH 7.4, 145 mM sodium ion).Hybridization conditions generally contain a monovalent cation andbiologically acceptable buffer and may or may not contain a divalentcation, complex anions, e.g. gluconate from potassium gluconate,uncharged species such as sucrose, and inert polymers to reduce theactivity of water in the sample, e.g. PEG. Such conditions includeconditions under which base pairs can form.

Hybridization is measured by the temperature required to dissociatesingle stranded nucleic acids forming a duplex, i.e., (the meltingtemperature; Tm). Hybridization conditions are also conditions underwhich base pairs can form. Various conditions of stringency can be usedto determine hybridization (see, e.g., Wahl, G. M. and S. L. Berger(1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol.152:507). Stringent temperature conditions will ordinarily includetemperatures of at least about 30° C., more preferably of at least about37° C., and most preferably of at least about 42° C. The hybridizationtemperature for hybrids anticipated to be less than 50 base pairs inlength should be 5-10° C. less than the melting temperature (Tm) of thehybrid, where Tm is determined according to the following equations. Forhybrids less than 18 base pairs in length, Tm(° C.)=2(# of A+Tbases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs inlength, Tm(° C.)=81.5+16.6(log 10[Na+])+0.41 (% G+C)−(600/N), where N isthe number of bases in the hybrid, and [Na+] is the concentration ofsodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M). Forexample, a hybridization determination buffer is shown in Table 1.

TABLE 1 To make 50 final conc. Vender Cat# Lot# m.w./Stock mL solutionNaCl 100 mM Sigma S-5150 41K8934   5M 1 mL KCl 80 mM Sigma P-954170K0002 74.55 0.298 g MgCl₂ 8 mM Sigma M-1028 120K8933   1M 0.4 mLsucrose 2% w/v Fisher BP220-212 907105 342.3 1 g Tris-HCl 16 mM FisherBP1757-500 12419   1M 0.8 mL NaH₂PO₄ 1 mM Sigma S-3193 52H-029515 120.00.006 g EDTA 0.02 mM Sigma E-7889 110K89271 0.5M 2 μL H₂O Sigma W-450251K2359 to 50 mL pH = 7.0 adjust with HCl at 20° C.

Useful variations on hybridization conditions will be readily apparentto those skilled in the art. Hybridization techniques are well known tothose skilled in the art and are described, for example, in Benton andDavis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad.Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in MolecularBiology, Wiley Interscience, New York, 2001); Berger and Kimmel(Antisense to Molecular Cloning Techniques, 1987, Academic Press, NewYork); and Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, New York.

As used herein, “oligonucleotide strand” is a single stranded nucleicacid molecule. An oligonucleotide may comprise ribonucleotides,deoxyribonucleotides, modified nucleotides (e.g., nucleotides with 2′modifications, synthetic base analogs, etc.) or combinations thereof.Such modified oligonucleotides can be preferred over native formsbecause of properties such as, for example, enhanced cellular uptake andincreased stability in the presence of nucleases.

Certain dsNAs of this invention are chimeric dsNAs. “Chimeric dsNAs” or“chimeras”, in the context of this invention, are dsNAs which containtwo or more chemically distinct regions, each made up of at least onenucleotide. These dsNAs typically contain at least one region primarilycomprising ribonucleotides (optionally including modifiedribonucleotides) that form a Dicer substrate siRNA (“DsiRNA”) molecule.This DsiRNA region is covalently attached, e.g., via conventionalphosphate bonds or via modified phosphate linkages (e.g.,phosphorothioate) to a second region comprising a single strandednucleotide region (“a single stranded extended region”) which confersone or more beneficial properties (such as, for example, increasedefficacy, e.g., increased potency and/or duration of DsiRNA activity,function as a recognition domain or means of targeting a chimeric dsNAto a specific location, for example, when administered to cells inculture or to a subject, functioning as an extended region for improvedattachment of functional groups, payloads, detection/detectablemoieties, functioning as an extended region that allows for moredesirable modifications and/or improved spacing of such modifications,etc.). This second region may also include modified or syntheticnucleotides and/or modified or synthetic deoxyribonucleotides.

As used herein, the term “ribonucleotide” encompasses natural andsynthetic, unmodified and modified ribonucleotides. Modificationsinclude changes to the sugar moiety, to the base moiety and/or to thelinkages between ribonucleotides in the oligonucleotide. As used herein,the term “ribonucleotide” specifically excludes a deoxyribonucleotide,which is a nucleotide possessing a single proton group at the 2′ ribosering position.

As used herein, the term “deoxyribonucleotide” encompasses natural andsynthetic, unmodified and modified deoxyribonucleotides. Modificationsinclude changes to the sugar moiety, to the base moiety and/or to thelinkages between deoxyribonucleotide in the oligonucleotide. As usedherein, the term “deoxyribonucleotide” also includes a modifiedribonucleotide that does not permit Dicer cleavage of a dsNA agent,e.g., a 2′-O-methyl ribonucleotide, a phosphorothioate-modifiedribonucleotide residue, etc., that does not permit Dicer cleavage tooccur at a bond of such a residue.

As used herein, the term “PS-NA” refers to a phosphorothioate-modifiednucleotide residue. The term “PS-NA” therefore encompasses bothphosphorothioate-modified ribonucleotides (“PS-RNAs”) andphosphorothioate-modified deoxyribonucleotides (“PS-DNAs”).

In certain embodiments, a chimeric DsiRNA/DNA agent of the inventioncomprises at least one duplex region of at least 23 nucleotides inlength, within which at least 50% of all nucleotides are unmodifiedribonucleotides. As used herein, the term “unmodified ribonucleotide”refers to a ribonucleotide possessing a hydroxyl (—OH) group at the 2′position of the ribose sugar.

In certain embodiments, a chimeric DsiRNA/DNA agent of the inventioncomprises at least one region, located 3′ of the projected Dicercleavage site on the first strand and 5′ of the projected Dicer cleavagesite on the second strand, having a length of at least 2 base pairednucleotides in length, wherein at least 50% of all nucleotides withinthis region of at least 2 base paired nucleotides in length areunmodified deoxyribonucleotides. As used herein, the term “unmodifieddeoxyribonucleotide” refers to a ribonucleotide possessing a singleproton at the 2′ position of the ribose sugar.

As used herein, antisense strand, guide strand and secondoligonucleotide refer to the same strand of a given dicer substratemolecule according to the invention; while sense strand, passengerstrand, and first oligonucleotide refer to the same strand of a givendicer substrate.

As used herein, “antisense strand” refers to a single stranded nucleicacid molecule which has a sequence complementary to that of a targetRNA. When the antisense strand contains modified nucleotides with baseanalogs, it is not necessarily complementary over its entire length, butmust at least hybridize with a target RNA

As used herein, “sense strand” refers to a single stranded nucleic acidmolecule which has a sequence complementary to that of an antisensestrand. When the antisense strand contains modified nucleotides withbase analogs, the sense strand need not be complementary over the entirelength of the antisense strand, but must at least be capable of forminga hybrid with, and thus be able to duplex with the antisense strand

As used herein, “guide strand” refers to a single stranded nucleic acidmolecule of a dsNA or dsNA-containing molecule, which has a sequencesufficiently complementary to that of a target RNA to result in RNAinterference. After cleavage of the dsNA or dsNA-containing molecule byDicer, a fragment of the guide strand remains associated with RISC,binds a target RNA as a component of the RISC complex, and promotescleavage of a target RNA by RISC. A guide strand is an antisense strand.

As used herein, “target RNA” refers to an RNA that would be subject tomodulation guided by the antisense strand, such as targeted cleavage orsteric blockage. The target RNA could be, for example genomic viral RNA,mRNA, a pre-mRNA, or a non-coding RNA. The preferred target is mRNA,such as the mRNA encoding a disease associated protein, such as ApoB,Bcl2, Hif-1alpha, Survivin or a p21 ras, such as Ha. ras, K-ras orN-ras.

As used herein, “passenger strand” refers to an oligonucleotide strandof a dsNA or dsNA-containing molecule, which has a sequence that iscomplementary to that of the guide strand A passenger strand is a sensestrand.

As used herein, “Dicer” refers to an endoribonuclease in the RNase IIIfamily that cleaves a dsRNA or dsRNA-containing molecule, e.g.,double-stranded RNA (dsRNA) or pre-microRNA (miRNA), intodouble-stranded nucleic acid fragments about 19-25 nucleotides long,usually with a two-base overhang on the 3′ end. With respect to thedsNAs of the invention, the duplex formed by a dsRNA region of a dsNA ofthe invention is recognized by Dicer and is a Dicer substrate on atleast one strand of the duplex. Dicer catalyzes the first step in theRNA interference pathway, which consequently results in the degradationof a target RNA. The protein sequence of human Dicer is provided at theNCBI database under accession number NP 085124, hereby incorporated byreference.

Dicer “cleavage” is determined as follows (e.g., see Collingwood et al.,Oligonucleotides 18:187-200 (2008)). In a Dicer cleavage assay, RNAduplexes (100 pmol) are incubated in 20 μL of 20 mM Tris pH 8.0, 200 mMNaCl, 2.5 mM MgCl2 with or without 1 unit of recombinant human Dicer(Stratagene, La Jolla, Calif.) at 37° C. for 18-24 hours. Samples aredesalted using a Performa SR 96-well plate (Edge Biosystems,Gaithersburg, Md.). Electrospray-ionization liquid chromatography massspectroscopy (ESI-LCMS) of duplex RNAs pre- and post-treatment withDicer is done using an Oligo HTCS system (Novatia, Princeton, N.J.; Hailet al., 2004), which consists of a ThermoFinnigan TSQ7000, Xcalibur datasystem, ProMass data processing software and Paradigm MS4 HPLC (MichromBioResources, Auburn, Calif.). In this assay, Dicer cleavage occurswhere at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, oreven 100% of the Dicer substrate dsRNA, (i.e., 25-35 bp dsRNA,preferably 26-30 bp dsRNA, optionally extended as described herein) iscleaved to a shorter dsRNA (e.g., 19-23 bp dsRNA, preferably, 21-23 bpdsRNA).

As used herein, “Dicer cleavage site” refers to the sites at which Dicercleaves a dsRNA (e.g., the dsRNA region of a dsNA of the invention).Dicer contains two RNase III domains which typically cleave both thesense and antisense strands of a dsRNA. The average distance between theRNase III domains and the PAZ domain determines the length of the shortdouble-stranded nucleic acid fragments it produces and this distance canvary (Macrae I, et al. (2006). “Structural basis for double-stranded RNAprocessing by Dicer”. Science 311 (5758): 195-8.). As shown, e.g., inFIG. 2, Dicer is projected to cleave certain double-stranded nucleicacids of the instant invention that possess an antisense strand having a2 nucleotide 3′ overhang at a site between the 21^(st) and 22^(nd)nucleotides removed from the 3′ terminus of the antisense strand, and ata corresponding site between the 21^(st) and 22^(nd) nucleotides removedfrom the 5′ terminus of the sense strand. The projected and/or prevalentDicer cleavage site(s) for dsNA molecules distinct from those depictedin FIG. 2 may be similarly identified via art-recognized methods,including those described in Macrae et al. While the Dicer cleavageevent depicted in FIG. 2 generates a 21 nucleotide siRNA, it is notedthat Dicer cleavage of a dsNA (e.g., DsiRNA) can result in generation ofDicer-processed siRNA lengths of 19 to 23 nucleotides in length. Indeed,in one aspect of the invention that is described in greater detailbelow, a double stranded DNA region is included within a dsNA forpurpose of directing prevalent Dicer excision of a typicallynon-preferred 19mer siRNA.

As used herein, “overhang” refers to unpaired nucleotides, in thecontext of a duplex having two or four free ends at either the 5′terminus or 3′ terminus of a dsNA. In certain embodiments, the overhangis a 3′ or 5′ overhang on the antisense strand or sense strand.

As used herein, “target” refers to any nucleic acid sequence whoseexpression or activity is to be modulated. In particular embodiments,the target refers to an RNA which duplexes to a single stranded nucleicacid that is an antisense strand in a RISC complex. Hybridization of thetarget RNA to the antisense strand results in processing by the RISCcomplex. Consequently, expression of the RNA or proteins encoded by theRNA, e.g., mRNA, is reduced.

As used herein, the term “RNA processing” refers to processingactivities performed by components of the siRNA, miRNA or RNase Hpathways (e.g., Drosha, Dicer, Argonaute2 or other RISCendoribonucleases, and RNaseH), which are described in greater detailbelow (see “RNA Processing” section below). The term is explicitlydistinguished from the post-transcriptional processes of 5′ capping ofRNA and degradation of RNA via non-RISC- or non-RNase H-mediatedprocesses. Such “degradation” of an RNA can take several forms, e.g.deadenylation (removal of a 3′ poly(A) tail), and/or nuclease digestionof part or all of the body of the RNA by any of several endo- orexo-nucleases (e.g., RNase III, RNase P, RNase T1, RNase A (1, 2, 3,4/5), oligonucleotidase, etc.).

As used herein, “reference” is meant a standard or control. As isapparent to one skilled in the art, an appropriate reference is whereonly one element is changed in order to determine the effect of the oneelement.

As used herein, “modified nucleotide” refers to a nucleotide that hasone or more modifications to the nucleoside, the nucleobase, furanosering, or phosphate group. For example, modified nucleotides excluderibonucleotides containing adenosine monophosphate, guanosinemonophosphate, uridine monophosphate, and cytidine monophosphate anddeoxyribonucleotides containing deoxyadenosine monophosphate,deoxyguanosine monophosphate, deoxythymidine monophosphate, anddeoxycytidine monophosphate. Modifications include those naturallyoccurring that result from modification by enzymes that modifynucleotides, such as methyltransferases. Modified nucleotides alsoinclude synthetic or non-naturally occurring nucleotides. Synthetic ornon-naturally occurring modifications in nucleotides include those with2′ modifications, e.g., 2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-O[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-(CH₂)₂—O-2′-bridge,2′-LNA, and 2′-O—(N-methylcarbamate) or those comprising base analogs.In connection with 2′-modified nucleotides as described for the presentdisclosure, by “amino” is meant 2′-NH₂ or 2′-O—NH₂, which can bemodified or unmodified. Such modified groups are described, e.g., inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S.Pat. No. 6,248,878.

The term “in vitro” has its art recognized meaning, e.g., involvingpurified reagents or extracts, e.g., cell extracts. The term “in vivo”also has its art recognized meaning, e.g., involving living cells, e.g.,immortalized cells, primary cells, cell lines, and/or cells in anorganism.

In reference to the nucleic acid molecules of the present disclosure,nucleotides in certain positions on either strand of the dsNA may bespecified. With reference to FIGS. 1-3, the conventions for denotingpositions of the DsiRNAs of the invention are shown in Table 2.

TABLE 2 Description of Numbering Convention as to Strand Positionsposition A position located on the passenger strand is denoted by anumber without a superscript label.(e.g., position 1). Position 1 of thepassenger strand is the 5′- terminal nucleotide, except for the 5′extended passenger strands, where the 5′ terminal nucleotide occurs inthe extended region and is accorded the highest number with asuperscript E (see below and FIG. 1A). position^(A) A position locatedon the guide strand is designated with a superscript A (e.g., position1^(A). The guide strand is numbered such that the first base pairednucleotide at its 3′ terminus is referred to as (e.g., position 1^(A)).Where the guide strand contains a 3′ terminal single stranded overhangof 1-6 nucleotides, those nucleotides are simply referred to as 3′terminal guide strand unpaired or single stranded residues. position^(B)A position located on the guide strand in the extended 5′ region islabeled with a superscript B (e.g., position 1^(B) represents the 5′terminal nucleotide of an extended guide strand (see FIG. 1A)).position^(C) A position located on the third oligonucleotide. The thirdoligonucleotide is complementary to the extended region of the guidestrand and is discontinuous with the passenger strand. Position 1^(C)(see FIG. 1A) represents the 5′ terminal nucleotide of the thirdoligoncleotide. position^(D) A position located on a 3′ extendedpassenger strand, such that position 1^(D) references the 3′ terminalnucleotide residue of the extended passenger strand position^(E) Aposition located on the extended region of a 5′ extended passengerstrand. Position 1^(E) is the unpaired nucleotide consecutive (i.e.,adjacent) to the first paired nucleotide of the passenger strand (seeFIG. 1A). position^(F) A position located in the duplex region of a 5′extended passenger strand, such that position 1^(F) references the firstpaired nucleotide on the 5′ passenger strand (starting from the 5′ end)and is the nucleotide consecutive to the position 1^(E) of the passengerstrand, which is an unpaired nucleotide of the strand 5′ single strandedextension.(see FIG. 1A).

In reference to the nucleic acid molecules of the present disclosure,the modifications may exist in patterns on a strand of the dsNA. As usedherein, “alternating positions” refers to a pattern where every othernucleotide is a modified nucleotide or there is an unmodified nucleotide(e.g., an unmodified ribonucleotide) between every modified nucleotideover a defined length of a strand of the dsNA (e.g., 5′-MNMNMN-3′;3′-MNMNMN-5′; where M is a modified nucleotide and N is an unmodifiednucleotide). The modification pattern starts from the first nucleotideposition at either the 5′ or 3′ terminus according to any of theposition numbering conventions described herein (in certain embodiments,position 1 is designated in reference to the terminal residue of astrand following a projected Dicer cleavage event of a DsiRNA agent ofthe invention; thus, position 1 does not always constitute a 3′ terminalor 5′ terminal residue of a pre-processed agent of the invention). Thepattern of modified nucleotides at alternating positions may run thefull length of the strand, but in certain embodiments includes at least4, 6, 8, 10, 12, 14 nucleotides containing at least 2, 3, 4, 5, 6 or 7modified nucleotides, respectively. As used herein, “alternating pairsof positions” refers to a pattern where two consecutive modifiednucleotides are separated by two consecutive unmodified nucleotides overa defined length of a strand of the dsNA (e.g., 5′-MMNNMMNNMMNN-3′;3′-MMNNMMNNMMNN-5′; where M is a modified nucleotide and N is anunmodified nucleotide). The modification pattern starts from the firstnucleotide position at either the 5′ or 3′ terminus according to any ofthe position numbering conventions described herein. The pattern ofmodified nucleotides at alternating positions may run the full length ofthe strand, but preferably includes at least 8, 12, 16, 20, 24, 28nucleotides containing at least 4, 6, 8, 10, 12 or 14 modifiednucleotides, respectively. It is emphasized that the above modificationpatterns are exemplary and are not intended as limitations on the scopeof the invention.

As used herein, “base analog” refers to a heterocyclic moiety which islocated at the 1′ position of a nucleotide sugar moiety in a modifiednucleotide that can be incorporated into a nucleic acid duplex (or theequivalent position in a nucleotide sugar moiety substitution that canbe incorporated into a nucleic acid duplex). In the dsNAs of theinvention, a base analog is generally either a purine or pyrimidine baseexcluding the common bases guanine (G), cytosine (C), adenine (A),thymine (T), and uracil (U). Base analogs can duplex with other bases orbase analogs in dsRNAs. Base analogs include those useful in thecompounds and methods of the invention., e.g., those disclosed in U.S.Pat. Nos. 5,432,272 and 6,001,983 to Benner and US Patent PublicationNo. 20080213891 to Manoharan, which are herein incorporated byreference. Non-limiting examples of bases include hypoxanthine (I),xanthine (X), 3β-D-ribofuranosyl-(2,6-diaminopyrimidine) (K),3-β-D-ribofuranosyl-(1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione)(P), iso-cytosine (iso-C), iso-guanine (iso-G),1-3-D-ribofuranosyl-(5-nitroindole),1-β-D-ribofuranosyl-(3-nitropyrrole), 5-bromouracil, 2-aminopurine,4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds) andpyrrole-2-carbaldehyde (Pa), 2-amino-6-(2-thienyl)purine (S),2-oxopyridine (Y), difluorotolyl, 4-fluoro-6-methylbenzimidazole,4-methylbenzimidazole, 3-methyl isocarbostyrilyl, 5-methylisocarbostyrilyl, and 3-methyl-7-propynyl isocarbostyrilyl,7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl,9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl,7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl,2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl,napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenzyl,tetracenyl, pentacenyl, and structural derivates thereof (Schweitzer etal., J. Org. Chem., 59:7238-7242 (1994); Berger et al., Nucleic AcidsResearch, 28(15):2911-2914 (2000); Moran et al., J. Am. Chem. Soc.,119:2056-2057 (1997); Morales et al., J. Am. Chem. Soc., 121:2323-2324(1999); Guckian et al., J. Am. Chem. Soc., 118:8182-8183 (1996); Moraleset al., J. Am. Chem. Soc., 122(6):1001-1007 (2000); McMinn et al., J.Am. Chem. Soc., 121:11585-11586 (1999); Guckian et al., J. Org. Chem.,63:9652-9656 (1998); Moran et al., Proc. Natl. Acad. Sci.,94:10506-10511 (1997); Das et al., J. Chem. Soc., Perkin Trans.,1:197-206 (2002); Shibata et al., J. Chem. Soc., Perkin Trans., 1:1605-1611 (2001); Wu et al., J. Am. Chem. Soc., 122(32):7621-7632(2000); O'Neill et al., J. Org. Chem., 67:5869-5875 (2002); Chaudhuri etal., J. Am. Chem. Soc., 117:10434-10442 (1995); and U.S. Pat. No.6,218,108.). Base analogs may also be a universal base.

As used herein, “universal base” refers to a heterocyclic moiety locatedat the 1′ position of a nucleotide sugar moiety in a modifiednucleotide, or the equivalent position in a nucleotide sugar moietysubstitution, that, when present in a nucleic acid duplex, can bepositioned opposite more than one type of base without altering thedouble helical structure (e.g., the structure of the phosphatebackbone). Additionally, the universal base does not destroy the abilityof the single stranded nucleic acid in which it resides to duplex to atarget nucleic acid. The ability of a single stranded nucleic acidcontaining a universal base to duplex a target nucleic can be assayed bymethods apparent to one in the art (e.g., UV absorbance, circulardichroism, gel shift, single stranded nuclease sensitivity, etc.).Additionally, conditions under which duplex formation is observed may bevaried to determine duplex stability or formation, e.g., temperature, asmelting temperature (Tm) correlates with the stability of nucleic acidduplexes. Compared to a reference single stranded nucleic acid that isexactly complementary to a target nucleic acid, the single strandednucleic acid containing a universal base forms a duplex with the targetnucleic acid that has a lower Tm than a duplex formed with thecomplementary nucleic acid. However, compared to a reference singlestranded nucleic acid in which the universal base has been replaced witha base to generate a single mismatch, the single stranded nucleic acidcontaining the universal base forms a duplex with the target nucleicacid that has a higher Tm than a duplex formed with the nucleic acidhaving the mismatched base.

Some universal bases are capable of base pairing by forming hydrogenbonds between the universal base and all of the bases guanine (G),cytosine (C), adenine (A), thymine (T), and uracil (U) under base pairforming conditions. A universal base is not a base that forms a basepair with only one single complementary base. In a duplex, a universalbase may form no hydrogen bonds, one hydrogen bond, or more than onehydrogen bond with each of G, C, A, T, and U opposite to it on theopposite strand of a duplex. Preferably, the universal bases does notinteract with the base opposite to it on the opposite strand of aduplex. In a duplex, base pairing between a universal base occurswithout altering the double helical structure of the phosphate backbone.A universal base may also interact with bases in adjacent nucleotides onthe same nucleic acid strand by stacking interactions. Such stackinginteractions stabilize the duplex, especially in situations where theuniversal base does not form any hydrogen bonds with the base positionedopposite to it on the opposite strand of the duplex. Non-limitingexamples of universal-binding nucleotides include inosine,1-13-D-ribofuranosyl-5-nitroindole, and/or1-β-D-ribofuranosyl-3-nitropyrrole (US Pat. Appl. Publ. No. 20070254362to Quay et al.; Van Aerschot et al., An acyclic 5-nitroindazolenucleoside analogue as ambiguous nucleoside. Nucleic Acids Res. 1995Nov. 11; 23(21):4363-70; Loakes et al., 3-Nitropyrrole and 5-nitroindoleas universal bases in primers for DNA sequencing and PCR. Nucleic AcidsRes. 1995 Jul. 11; 23(13):2361-6; Loakes and Brown, 5-Nitroindole as anuniversal base analogue. Nucleic Acids Res. 1994 Oct. 11;22(20):4039-43).

As used herein, “loop” refers to a structure formed by a single strandof a nucleic acid, in which complementary regions that flank aparticular single stranded nucleotide region hybridize in a way that thesingle stranded nucleotide region between the complementary regions isexcluded from duplex formation or Watson-Crick base pairing. A loop is asingle stranded nucleotide region of any length. Examples of loopsinclude the unpaired nucleotides present in such structures as hairpins,stem loops, or extended loops.

As used herein, “extended loop” in the context of a dsRNA refers to asingle stranded loop and in addition 1, 2, 3, 4, 5, 6 or up to 20 basepairs or duplexes flanking the loop. In an extended loop, nucleotidesthat flank the loop on the 5′ side form a duplex with nucleotides thatflank the loop on the 3′ side. An extended loop may form a hairpin orstem loop.

As used herein, “tetraloop” in the context of a dsRNA refers to a loop(a single stranded region) consisting of four nucleotides that forms astable secondary structure that contributes to the stability of anadjacent Watson-Crick hybridized nucleotides. Without being limited totheory, a tetraloop may stabilize an adjacent Watson-Crick base pair bystacking interactions. In addition, interactions among the fournucleotides in a tetraloop include but are not limited tonon-Watson-Crick base pairing, stacking interactions, hydrogen bonding,and contact interactions (Cheong et al., Nature 1990 Aug. 16;346(6285):680-2; Heus and Pardi, Science 1991 Jul. 12; 253(5016):191-4).A tetraloop confers an increase in the melting temperature (Tm) of anadjacent duplex that is higher than expected from a simple model loopsequence consisting of four random bases. For example, a tetraloop canconfer a melting temperature of at least 55° C. in 10 mM NaHPO₄ to ahairpin comprising a duplex of at least 2 base pairs in length. Atetraloop may contain ribonucleotides, deoxyribonucleotides, modifiednucleotides, and combinations thereof. Examples of RNA tetraloopsinclude the UNCG family of tetraloops (e.g., UUCG), the GNRA family oftetraloops (e.g., GAAA), and the CUUG tetraloop. (Woese et al., ProcNatl Acad Sci USA. 1990 November; 87(21):8467-71; Antao et al., NucleicAcids Res. 1991 Nov. 11; 19(21):5901-5). Examples of DNA tetraloopsinclude the d(GNNA) family of tetraloops (e.g., d(GTTA), the d(GNRA))family of tetraloops, the d(GNAB) family of tetraloops, the d(CNNG)family of tetraloops, the d(TNCG) family of tetraloops (e.g., d(TTCG)).(Nakano et al. Biochemistry, 41 (48), 14281-14292, 2002.; SHINJI et al.Nippon Kagakkai Koen Yokoshu VOL.78th; NO.2; PAGE.731 (2000).)

As used herein, “increase” or “enhance” is meant to alter positively byat least 5% compared to a reference in an assay. An alteration may be by5%, 10%, 25%, 30%, 50%, 75%, or even by 100% compared to a reference inan assay. By “enhance Dicer cleavage,” it is meant that the processingof a quantity of a dsRNA or dsRNA-containing molecule by Dicer resultsin more Dicer cleaved dsRNA products, that Dicer cleavage reactionoccurs more quickly compared to the processing of the same quantity of areference dsRNA or dsRNA-containing molecule in an in vivo or in vitroassay of this disclosure, or that Dicer cleavage is directed to cleaveat a specific, preferred site within a dsNA and/or generate higherprevalence of a preferred population of cleavage products (e.g., byinclusion of DNA residues as described herein). In one embodiment,enhanced or increased Dicer cleavage of a dsNA molecule is above thelevel of that observed with an appropriate reference dsNA molecule. Inanother embodiment, enhanced or increased Dicer cleavage of a dsNAmolecule is above the level of that observed with an inactive orattenuated molecule.

As used herein “reduce” is meant to alter negatively by at least 5%compared to a reference in an assay. An alteration may be by 5%, 10%,25%, 30%, 50%, 75%, or even by 100% compared to a reference in an assay.By “reduce expression,” it is meant that the expression of the gene, orlevel of RNA molecules or equivalent RNA molecules encoding one or moreproteins or protein subunits, or level or activity of one or moreproteins or protein subunits encoded by a target gene, is reduced belowthat observed in the absence of the nucleic acid molecules (e.g., dsRNAmolecule or dsRNA-containing molecule) in an in vivo or in vitro assayof this disclosure. In one embodiment, inhibition, down-regulation orreduction with a dsNA molecule is below that level observed in thepresence of an inactive or attenuated molecule. In another embodiment,inhibition, down-regulation, or reduction with dsNA molecules is belowthat level observed in the presence of, e.g., a dsNA molecule withscrambled sequence or with mismatches. In another embodiment,inhibition, down-regulation, or reduction of gene expression with anucleic acid molecule of the instant disclosure is greater in thepresence of the nucleic acid molecule than in its absence.

As used herein, “cell” is meant to include both prokaryotic (e.g.,bacterial) and eukaryotic (e.g., mammalian or plant) cells. Cells may beof somatic or germ line origin, may be totipotent or pluripotent, andmay be dividing or non-dividing. Cells can also be derived from or cancomprise a gamete or an embryo, a stem cell, or a fully differentiatedcell. Thus, the term “cell” is meant to retain its usual biologicalmeaning and can be present in any organism such as, for example, a bird,a plant, and a mammal, including, for example, a human, a cow, a sheep,an ape, a monkey, a pig, a dog, and a cat. Within certain aspects, theterm “cell” refers specifically to mammalian cells, such as human cells,that contain one or more isolated dsNA molecules of the presentdisclosure. In particular aspects, a cell processes dsRNAs ordsRNA-containing molecules resulting in RNA interference of targetnucleic acids, and contains proteins and protein complexes required forRNAi, e.g., Dicer and RISC.

As used herein, “animal” is meant a multicellular, eukaryotic organism,including a mammal, particularly a human. The methods of the inventionin general comprise administration of an effective amount of the agentsherein, such as an agent of the structures of formulae herein, to asubject (e.g., animal, human) in need thereof, including a mammal,particularly a human. Such treatment will be suitably administered tosubjects, particularly humans, suffering from, having, susceptible to,or at risk for a disease, or a symptom thereof.

By “pharmaceutically acceptable carrier” is meant, a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant disclosure in the physical location mostsuitable for their desired activity.

The present invention is directed to compositions that comprise both adouble stranded RNA (“dsRNA”) duplex and DNA-containing extendedregion—in most embodiments, a dsDNA duplex—within the same agent, andmethods for preparing them, that are capable of reducing the expressionof target genes in eukaryotic cells. One of the strands of the dsRNAregion contains a region of nucleotide sequence that has a length thatranges from about 15 to about 22 nucleotides that can direct thedestruction of the RNA transcribed from the target gene. The dsDNAduplex region of such an agent is not necessarily complementary to thetarget RNA, and, therefore, in such instances does not enhance targetRNA hybridization of the region of nucleotide sequence capable ofdirecting destruction of a target RNA. Double stranded NAs of theinvention can possess strands that are chemically linked, or can alsopossess an extended loop, optionally comprising a tetraloop, that linksthe first and second strands. In some embodiments, the extended loopcontaining the tetraloop is at the 3′ terminus of the sense strand, atthe 5′ terminus of the antisense strand, or both.

In one embodiment, the dsNA of the invention comprises a double strandedRNA duplex region comprising 18-30 nts (for example, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29 and 30 nts) in length.

“Extended” DsiRNA agents according to the invention can be categorizedas either “guide extended” (the nucleotide region at the 5′ terminus ofthe antisense strand that is present on the molecule in addition to the26-30 base antisense sequence required for participation of theantisense strand in a dicer substrate) or “passenger extended” (thenucleotide region at the 3′ terminus of the sense strand that isoptionally present on the molecule in addition to the 25-30 base sensesequence required for sense strand participation in a dicer substrate;or the nucleotide region at the 5′ terminus of the sense strand that isoptionally present on the sense strand in addition to the 25-30 basesequence required for sense strand participation in a dicer substrate).Therefore, as used herein, the term “extended” is not meant to refer tothe antisense (or second strand, or guide strand) 3′ overhang of 1-6single stranded nucleotides; rather “extended” as used herein refers tothe opposite end of the dicer substrate molecule, that is, a 5′ extendedantisense strand, where the extended region is 10-30, preferably 10-15nucleotides in length or a 3′ extended sense strand, where the extendedregion is 10-30, preferably 10-15 nucleotides in length. The 5′ extendedantisense strand may be single stranded, and optionally may be duplexedwith a third nucleic acid molecule which is complementary, preferablyfully (100%) complementary, to the 5′ extended single stranded region ofthe antisense strand. Therefore, in some embodiments, i.e., when thethird nucleic acid molecule is present, the 5′ extended region of theantisense strand is not single stranded, but rather is a duplex, ordouble stranded region. Preferably, according to the invention, thethird nucleic acid molecule, i.e., the sense region that iscomplementary to the 5′ extended antisense region, is not present unlessa cognate 5′ extended antisense region is present.

The DsiRNA/dsDNA agents of the instant invention can enhance thefollowing attributes of such agents relative to DsiRNAs lacking extendedsecond strand (e.g., antisense) 5′ regions or extended first strand(e.g., sense) 3′ regions: in vitro efficacy (e.g., potency and durationof effect), in vivo efficacy (e.g., potency, duration of effect,pharmacokinetics, pharmacodynamics, intracellular uptake, reducedtoxicity). In certain embodiments, the 5′ extended region of the secondstrand or 3′ extended region of the first strand can optionally providean additional agent (or fragment thereof), such as an aptamer orfragment thereof; a binding site (e.g., a “decoy” binding site) for anative or exogenously introduced moiety capable of binding to a 5′extended second strand nucleotide region or 3′ extended first strandregion, respectively in either a non-sequence-selective orsequence-specific manner (e.g., the 5′ extended second strand nucleotideregion of an agent of the instant invention can be designed to compriseone or more transcription factor recognition sequences and/or the 5′extended second strand nucleotide region can provide a sequence-specificrecognition domain for a probe, marker, etc.).

As used herein, the term “pharmacokinetics” refers to the process bywhich a drug is absorbed, distributed, metabolized, and eliminated bythe body. In certain embodiments of the instant invention, enhancedpharmacokinetics of a 5′ extended second strand pr 3′ extended firststrand DsiRNA agent relative to an appropriate control DsiRNA refers toincreased absorption and/or distribution of such an agent, and/or slowedmetabolism and/or elimination of such a 5′ second strand extended DsiRNAagent or 3′ first strand extended DsiRNA agent from a subjectadministered such an agent.

As used herein, the term “pharmacodynamics” refers to the action oreffect of a drug on a living organism. In certain embodiments of theinstant invention, enhanced pharmacodynamics of a 5′ second strandextended DsiRNA agent or 3′ first strand extended DsiRNA agent relativeto an appropriate control DsiRNA refers to an increased (e.g., morepotent or more prolonged) action or effect of a 5′ second strandextended DsiRNA agent or 3′ first strand extended DsiRNA agent,respectively, upon a subject administered such agent, relative to anappropriate control DsiRNA.

As used herein, the term “stabilization” refers to a state of enhancedpersistence of an agent in a selected environment (e.g., in a cell ororganism). In certain embodiments, the 5′ second strand extended DsiRNAor 3′ first strand extended DsiRNA agents of the instant inventionexhibit enhanced stability relative to appropriate control DsiRNAs. Suchenhanced stability can be achieved via enhanced resistance of suchagents to degrading enzymes (e.g., nucleases) or other agents.

In addition to the attributes described above for the 5′ antisenseextended dicer substrates according to the invention, where the optionalthird nucleic acid sense molecule of 10-30, preferably 10-15 nucleotidesis present in a molecule, this third sense molecule may function tostabilize the entire molecule, and/or to confer another advantage, suchas increase potency, prolong action or effect, enhance pharmacodynamicor pharmacological effects, and/or to provide an additional agent (orportion thereof), such as an aptamer or fragment thereof; a binding site(e.g., a “decoy” binding site) for a native or exogenously introducedmoiety (e.g., a label) that is bound to and thus carried by the thirdmolecule as it participates in the dicer substrate.

DsiRNA Design/Synthesis

It was previously shown that longer dsRNA species of from 25 to about 30nucleotides (DsiRNAs) yield unexpectedly effective RNA inhibitoryresults in terms of potency and duration of action, as compared to19-23mer siRNA agents. Without wishing to be bound by the underlyingtheory of the dsRNA processing mechanism, it is thought that the longerdsRNA species serve as a substrate for the Dicer enzyme in the cytoplasmof a cell. In addition to cleaving the dsNA of the invention intoshorter segments, Dicer is thought to facilitate the incorporation of asingle-stranded cleavage product derived from the cleaved dsNA into theRISC complex that is responsible for the destruction of the cytoplasmicRNA of or derived from the target gene. Prior studies (Rossi et al.,U.S. Patent Application No. 2007/0265220) have shown that thecleavability of a dsRNA species (specifically, a DsiRNA agent) by Dicercorresponds with increased potency and duration of action of the dsRNAspecies. The instant invention, at least in part, provides for design ofRNA inhibitory agents that direct the site of Dicer cleavage, such thatpreferred species of Dicer cleavage products are thereby generated.

In a model of DsiRNA processing, Dicer enzyme binds to a DsiRNA agent,resulting in cleavage of the DsiRNA at a position 19-23 nucleotidesremoved from a Dicer PAZ domain-associated 3′ overhang sequence of theantisense strand of the DsiRNA agent. This Dicer cleavage event resultsin excision of those duplexed nucleic acids previously located at the 3′end of the passenger (sense) strand and 5′ end of the guide (antisense)strand. Cleavage of a DsiRNA typically yields a 19mer duplex with 2-baseoverhangs at each end. As presently modeled in FIG. 2, this Dicercleavage event generates a 21-23 nucleotide guide (antisense) strand(or, in certain instances where a longer guide strand 3′ overhang ispresent, 24-27 nucleotide guide strands could result from Dicercleavage) capable of directing sequence-specific inhibition of targetmRNA as a RISC component.

The first and second oligonucleotides of the DsiRNA agents of theinstant invention are not required to be completely complementary in theduplexed region. In one embodiment, the 3′-terminus of the sense strandcontains one or more mismatches. In one aspect, about two mismatches areincorporated at the 3′ terminus of the sense strand. In anotherembodiment, the DsiRNA of the invention is a double stranded RNAmolecule containing two RNA oligonucleotides in the range of 25-66nucleotides in length and, when annealed to each other, have a twonucleotide mismatch on the 3′-terminus of the sense strand (the5′-terminus of the antisense strand). The use of mismatches or decreasedthermodynamic stability (specifically at the 3′-sense/5′-antisenseposition) has been proposed to facilitate or favor entry of theantisense strand into RISC (Schwarz et al., 2003; Khvorova et al.,2003), presumably by affecting some rate-limiting unwinding steps thatoccur with entry of the siRNA into RISC. Thus, terminal base compositionhas been included in design algorithms for selecting active 21mer siRNAduplexes (Ui-Tei et al., 2004; Reynolds et al., 2004). With Dicercleavage of the dsRNA region of this embodiment, the small end-terminalsequence which contains the mismatches will either be left unpaired withthe antisense strand (become part of a 3′-overhang) or be cleavedentirely off the final 21-mer siRNA. These specific forms of“mismatches”, therefore, do not persist as mismatches in the final RNAcomponent of RISC. The finding that base mismatches or destabilizationof segments at the 3′-end of the sense strand of Dicer substrateimproved the potency of synthetic duplexes in RNAi, presumably byfacilitating processing by Dicer, was a surprising finding of past worksdescribing the design and use of 25-30mer dsRNAs (also termed “DsiRNAs”herein; Rossi et al., U.S. Patent Application Nos. 2005/0277610,2005/0244858 and 2007/0265220). Exemplary mismatched or wobble basepairs of agents possessing mismatches are G:A, C:A, C:U, G:G, A:A, C:C,U:U, I:A, I:U and I:C. Base pair strength of such agents can also belessened via modification of the nucleotides of such agents, including,e.g., 2-amino- or 2,6-diamino modifications of guanine and adeninenucleotides.

Exemplary Structures of DsiRNA Agent Compositions

The compositions of the invention comprise a dsNA which is a precursormolecule, i.e., the dsNA of the present invention is processed in vivoto produce an active small interfering nucleic acid (siRNA). The dsNA isprocessed by Dicer to an active siRNA which is incorporated into RISC.

In one aspect, the present invention provides compositions for RNAinterference (RNAi) having a first or second strand that has at least 8contiguous ribonucleotides. In certain embodiments, a DsiRNA of theinvention has 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23or more (e.g., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 26, ormore, up to the full length of the strand) ribonucleotides, modifiedribonucleotides (2′-O-methyl ribonucleotides, phosphorothioatelinkages). In certain embodiments, the ribonucleotides or modifiedribonucleotides are contiguous.

In one aspect, the present invention provides compositions for RNAinterference (RNAi) that possess one or more deoxyribonucleotides withina region of a double stranded nucleic acid that is positioned 3′ of aprojected sense strand Dicer cleavage site and correspondingly 5′ of aprojected antisense strand Dicer cleavage site. In one embodiment, atleast one nucleotide of the guide strand between and including the guidestrand nucleotides corresponding to and thus base paired with passengerstrand positions 24 to the 3′ terminal nucleotide residue of thepassenger strand is a deoxyribonucleotide. In some embodiments, thedouble stranded nucleic acid possesses one or more base paireddeoxyribonucleotides within a region of the double stranded nucleic acidthat is positioned 3′ of a projected sense strand Dicer cleavage siteand correspondingly 5′ of a projected antisense strand Dicer cleavagesite

In certain embodiments, the DsiRNA agents of the invention can have anyof the following exemplary structures:

In one such embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX_(N*)-3′3′-YXXXXXXXXXXXXXXXXXXXXXXXXX_(N*)Z_(N)-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 0-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “Z”=DNA, RNA, or modifiednucleotide, and “N”=1 to 50 or more, but is optionally 1-30 or,optionally 1-15 or, optionally, 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, or 5. In one embodiment, the top strand is thesense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand.

In a related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXX_(N*)DD- 3′3′-YXXXXXXXXXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “D”=DNA, “Z”=DNA, RNA, ormodified nucleotide, and “N”=1 to 50 or more, but is optionally 1-30 or,optionally 1-15 or, optionally, 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, or 5. In one embodiment, the top strand is thesense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand.

In another such embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX_(N*)|E_(N)-3′3′-YXXXXXXXXXXXXXXXXXXXXXXXXX_(N*)Z_(N)-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 0-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “Z”=DNA, RNA, or modifiednucleotide, and “N”=1 to 50 or more, but is optionally 1-30 or,optionally 1-15 or, optionally, 1-10. “E”=DNA, RNA, or modifiednucleotide, “1”=a discontinuity, and “N”=1 to 50 or more, but isoptionally 1-15 or, optionally, 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, or 5. In one embodiment, the top strand is thesense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand.

In a related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXX_(N*)DD|E_(N)-3′3′-YXXXXXXXXXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “D”=DNA, “Z”=DNA, RNA, ormodified nucleotide, and “N”=1 to 50 or more, but is optionally 1-30 or,optionally 1-15 or, optionally, 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, or 5. In one embodiment, the top strand is thesense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand.

In another such embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX_(N*)Z_(N)-3′3′-YXXXXXXXXXXXXXXXXXXXXXXXXX_(N*)-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “Z”=DNA, RNA, or modifiednucleotide, and “N”=1 to 50 or more, but is optionally 1-30 or,optionally 1-15 or, optionally, 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, or 5. In one embodiment, the top strand is thesense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand.

In a related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXX_(N*)DDZ_(N)-3′3′-YXXXXXXXXXXXXXXXXXXXXXXX_(N*)XX-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “D”=DNA, “Z”=DNA, RNA, ormodified nucleotide, and “N”=1 to 50 or more, but is optionally 1-30 or,optionally 1-15 or, optionally, 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, or 5. In one embodiment, the top strand is thesense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand.

In an additional embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXX_(N*)DDZ_(N)-3′3′-YXXXXXXXXXXXXXXXXXXXXXXX_(N*)XX-5′wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an optional overhang domaincomprised of 0-10 RNA monomers that are optionally 2′-O-methyl RNAmonomers—in certain embodiments, “Y” is an overhang domain comprised of1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, “D”=DNA,“Z”=DNA, RNA, or modified nucleotide, and “N”=1 to 50 or more, but isoptionally 1-30 or, optionally 1-15 or, optionally, 1-10. “N*”=0 to 15or more, but is optionally 0, 1, 2, 3, 4, or 5. In one embodiment, thetop strand is the sense strand, and the bottom strand is the antisensestrand. Alternatively, the bottom strand is the sense strand and the topstrand is the antisense strand.

In another such embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX_(N*)-3′3′-YXXXXXXXXXXXXXXXXXXXXXXXXX_(N*)Z_(N)-5′wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an optional overhang domaincomprised of 0-10 RNA monomers that are optionally 2′-O-methyl RNAmonomers—in certain embodiments, “Y” is an overhang domain comprised of1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, “Z”=DNA,RNA, or modified nucleotide, and “N”=1 to 50 or more, but is optionally1-30 or, optionally 1-15 or, optionally, 1-10. “N*”=0 to 15 or more, butis optionally 0, 1, 2, 3, 4, or 5. In one embodiment, the top strand isthe sense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand. In one embodiment, the top strand is the sensestrand, and the bottom strand is the antisense strand. Alternatively,the bottom strand is the sense strand and the top strand is theantisense strand, with 2′-O-methyl RNA monomers located at alternatingresidues along the top strand, rather than the bottom strand presentlydepicted in the above schematic.

In another such embodiment, the DsiRNA comprises:

5'-XXXXXXXXXXXXXXXXXXXXXXX_(N*)DD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an optional overhang domaincomprised of 0-10 RNA monomers that are optionally 2′-O-methyl RNAmonomers—in certain embodiments, “Y” is an overhang domain comprised of1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, “D”=DNA,“Z”=DNA, RNA, or modified nucleotide, and “N”=1 to 50 or more, but isoptionally 1-30 or, optionally 1-15 or, optionally, 1-10. “N*”=0 to 15or more, but is optionally 0, 1, 2, 3, 4, or 5. In one embodiment, thetop strand is the sense strand, and the bottom strand is the antisensestrand. Alternatively, the bottom strand is the sense strand and the topstrand is the antisense strand.

In one embodiment, the top strand is the sense strand, and the bottomstrand is the antisense strand. Alternatively, the bottom strand is thesense strand and the top strand is the antisense strand, with2′-O-methyl RNA monomers located at alternating residues along the topstrand, rather than the bottom strand presently depicted in the aboveschematic.

In any of the above-depicted structures, the 5′ end of either the sensestrand or antisense strand optionally comprises a phosphate group.

In another such embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX_(N*)|E_(N)-3′3′-YXXXXXXXXXXXXXXXXXXXXXXXXX_(N*)Z_(N)-5′wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an optional overhang domaincomprised of 0-10 RNA monomers that are optionally 2′-O-methyl RNAmonomers—in certain embodiments, “Y” is an overhang domain comprised of1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, “Z”=DNA,RNA, or modified nucleotide, and “N”=1 to 50 or more, but is optionally1-30 or, optionally 1-15 or, optionally, 1-10. “E”=DNA, RNA, or modifiednucleotide, “1”=a discontinuity, and “N”=1 to 50 or more, but isoptionally 1-15 or, optionally, 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, or 5. In one embodiment, the top strand is thesense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand. In one embodiment, the top strand is the sensestrand, and the bottom strand is the antisense strand. Alternatively,the bottom strand is the sense strand and the top strand is theantisense strand, with 2′-O-methyl RNA monomers located at alternatingresidues along the top strand, rather than the bottom strand presentlydepicted in the above schematic.

In a related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXX_(N*)DD|E_(N)-3′3′-YXXXXXXXXXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′

wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an optional overhang domaincomprised of 0-10 RNA monomers that are optionally 2′-O-methyl RNAmonomers—in certain embodiments, “Y” is an overhang domain comprised of1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, “D”=DNA,“Z”=DNA, RNA, or modified nucleotide, and “N”=1 to 50 or more, but isoptionally 1-30 or, optionally 1-15 or, optionally, 1-10. “N*”=0 to 15or more, but is optionally 0, 1, 2, 3, 4, or 5. In one embodiment, thetop strand is the sense strand, and the bottom strand is the antisensestrand. Alternatively, the bottom strand is the sense strand and the topstrand is the antisense strand. In one embodiment, the top strand is thesense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand, with 2′-O-methyl RNA monomers located atalternating residues along the top strand, rather than the bottom strandpresently depicted in the above schematic.

In another such embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXXXX_(N*)Z_(N)-3′3′-YXXXXXXXXXXXXXXXXXXXXXXXXX_(N*)-5′wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an optional overhang domaincomprised of 0-10 RNA monomers that are optionally 2′-O-methyl RNAmonomers—in certain embodiments, “Y” is an overhang domain comprised of1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, “Z”=DNA,RNA, or modified nucleotide, and “N”=1 to 50 or more, but is optionally1-30 or, optionally 1-15 or, optionally, 1-10. “N*”=0 to 15 or more, butis optionally 0, 1, 2, 3, 4, or 5. In one embodiment, the top strand isthe sense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand. In one embodiment, the top strand is the sensestrand, and the bottom strand is the antisense strand. Alternatively,the bottom strand is the sense strand and the top strand is theantisense strand, with 2′-O-methyl RNA monomers located at alternatingresidues along the top strand, rather than the bottom strand presentlydepicted in the above schematic.

In a related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXXXX_(N*)DDZ_(N)-3′3′-YXXXXXXXXXXXXXXXXXXXXXXX_(N*)XX-5′wherein “X”=RNA, “X”=2′-O-methyl RNA, “Y” is an optional overhang domaincomprised of 0-10 RNA monomers that are optionally 2′-O-methyl RNAmonomers—in certain embodiments, “Y” is an overhang domain comprised of1-4 RNA monomers that are optionally 2′-O-methyl RNA monomers, “D”=DNA,“Z”=DNA, RNA, or modified nucleotide, and “N”=1 to 50 or more, but isoptionally 1-30 or, optionally 1-15 or, optionally, 1-10. “N*”=0 to 15or more, but is optionally 0, 1, 2, 3, 4, or 5. In one embodiment, thetop strand is the sense strand, and the bottom strand is the antisensestrand. Alternatively, the bottom strand is the sense strand and the topstrand is the antisense strand. In one embodiment, the top strand is thesense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand, with 2′-O-methyl RNA monomers located atalternating residues along the top strand, rather than the bottom strandpresently depicted in the above schematic.

In one embodiment, a extended DsiRNA agent is provided that comprisesdeoxyribonucleotides positioned at sites modeled to function viaspecific direction of Dicer cleavage. An exemplary structure for such amolecule is shown:

5′-XXXXXXXXXXXXXXXXXXXXX_(N*)XXDD-3′3′-YXXXXXXXXXXXXXXXXXXXXX_(N*)DDXXZ_(N)-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “D”=DNA, “Z”=DNA, RNA, ormodified nucleotide, and “N”=1 to 50 or more, but is optionally 1-30 or,optionally 1-15 or, optionally, 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, or 5. In one embodiment, the top strand is thesense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand.

The above structure is modeled to force Dicer to cleave a maximum of a21mer duplex as its primary post-processing form. In embodiments wherethe bottom strand of the above structure is the antisense strand, thepositioning of two deoxyribonucleotide residues at the ultimate andpenultimate residues of the 5′ end of the antisense strand is likely toreduce off-target effects (as prior studies have shown a 2′-O-methylmodification of at least the penultimate position from the 5′ terminusof the antisense strand to reduce off-target effects; see, e.g., US2007/0223427).

In a related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXX_(N*)XXDD|E_(N)-3′3′-YXXXXXXXXXXXXXXXXXXXXX_(N*)DDXXZ_(N)-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “D”=DNA, “Z”=DNA, RNA, ormodified nucleotide, and “N”=1 to 50 or more, but is optionally 1-30 or,optionally 1-15 or, optionally, 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, or 5. In one embodiment, the top strand is thesense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand.

In a related embodiment, the DsiRNA comprises:

5′-XXXXXXXXXXXXXXXXXXXXX_(N*)XXDDZ_(N)-3′3′-YXXXXXXXXXXXXXXXXXXXXX_(N*)DDXX-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “D”=DNA, “Z”=DNA, RNA, ormodified nucleotide, and “N”=1 to 50 or more, but is optionally 1-30 or,optionally 1-15 or, optionally, 1-10. “N*”=0 to 15 or more, but isoptionally 0, 1, 2, 3, 4, or 5. In one embodiment, the top strand is thesense strand, and the bottom strand is the antisense strand.Alternatively, the bottom strand is the sense strand and the top strandis the antisense strand.

In any of the above-depicted structures, the 5′ end of either the sensestrand or antisense strand optionally comprises a phosphate group.

In one embodiment, the present invention provides a double strandednucleic acid having a substantially duplexed region between the firstand second strands comprising a fully duplexed region having no unpairedbases between the 5′ terminal and 3′ terminal nucleotides of the firststrand that are paired with corresponding nucleotides of the secondstrand. In another embodiment, the present invention provides a doublestranded nucleic acid having a substantially duplexed region comprising,between the 5′ terminal and 3′ terminal nucleotides of the first strandthat are paired with corresponding nucleotides of the second strand, 1unpaired base pair, 2 unpaired base pairs, 3 unpaired base pairs, 4unpaired base pairs, and 5 unpaired base pairs. In some embodiments, theunpaired base pairs are consecutive. In other embodiments, the unpairedbase pairs are non-consecutive.

As used herein “DsiRNAmm” refers to a DsiRNA having a “mismatch tolerantregion” containing one, two, three or four mismatched base pairs of theduplex formed by the sense and antisense strands of the DsiRNA, wheresuch mismatches are positioned within the DsiRNA at a location(s) lyingbetween (and thus not including) the two terminal base pairs of eitherend of the double stranded region of the DsiRNA. The mismatched basepairs are located within a “mismatch-tolerant region” which is definedherein with respect to the location of the projected Ago2 cut site ofthe corresponding target nucleic acid. The mismatch tolerant region islocated “upstream of” the projected Ago2 cut site of the target strand.“Upstream” in this context will be understood as the 5′-most portion ofthe DsiRNAmm duplex, where 5′ refers to the orientation of the sensestrand of the DsiRNA duplex. Therefore, the mismatch tolerant region isupstream of the base on the sense (passenger) strand that corresponds tothe projected Ago2 cut site of the target nucleic acid; alternatively,when referring to the antisense (guide) strand of the DsiRNAmm, themismatch tolerant region can also be described as positioned downstreamof the base that is complementary to the projected Ago2 cut site of thetarget nucleic acid, that is, the 3′-most portion of the antisensestrand of the DsiRNAmm (where position 1 of the antisense strand is the5′ terminal nucleotide of the antisense strand).

In one embodiment, for example, the mismatch tolerant region ispositioned between and including base pairs 3-9 when numbered from thenucleotide starting at the 5′ end of the sense strand of the duplex.Therefore, a DsiRNAmm of the invention possesses a single mismatchedbase pair at any one of positions 3, 4, 5, 6, 7, 8 or 9 of the sensestrand of a right-hand extended DsiRNA (where position 1 is the 5′terminal nucleotide of the sense strand and position 9 is the nucleotideresidue of the sense strand that is immediately 5′ of the projected Ago2cut site of the target RNA sequence corresponding to the sense strandsequence). In certain embodiments, for a DsiRNAmm that possesses amismatched base pair nucleotide at any of positions 3, 4, 5, 6, 7, 8 or9 of the sense strand, the corresponding mismatched base pair nucleotideof the antisense strand not only forms a mismatched base pair with theDsiRNAmm sense strand sequence, but also forms a mismatched base pairwith a DsiRNAmm target RNA sequence (thus, complementarity between theantisense strand sequence and the sense strand sequence is disrupted atthe mismatched base pair within the DsiRNAmm, and complementarity issimilarly disrupted between the antisense strand sequence of theDsiRNAmm and the target RNA sequence). In alternative embodiments, themismatch base pair nucleotide of the antisense strand of a DsiRNAmm onlyform a mismatched base pair with a corresponding nucleotide of the sensestrand sequence of the DsiRNAmm, yet base pairs with its correspondingtarget RNA sequence nucleotide (thus, complementarity between theantisense strand sequence and the sense strand sequence is disrupted atthe mismatched base pair within the DsiRNAmm, yet complementarity ismaintained between the antisense strand sequence of the DsiRNAmm and thetarget RNA sequence).

A DsiRNAmm of the invention that possesses a single mismatched base pairwithin the mismatch-tolerant region (mismatch region) as described above(e.g., a DsiRNAmm harboring a mismatched nucleotide residue at any oneof positions 3, 4, 5, 6, 7, 8 or 9 of the sense strand) can furtherinclude one, two or even three additional mismatched base pairs. Inpreferred embodiments, these one, two or three additional mismatchedbase pairs of the DsiRNAmm occur at position(s) 3, 4, 5, 6, 7, 8 and/or9 of the sense strand (and at corresponding residues of the antisensestrand). In one embodiment where one additional mismatched base pair ispresent within a DsiRNAmm, the two mismatched base pairs of the sensestrand can occur, e.g., at nucleotides of both position 4 and position 6of the sense strand (with mismatch also occurring at correspondingnucleotide residues of the antisense strand).

In DsiRNAmm agents possessing two mismatched base pairs, mismatches canoccur consecutively (e.g., at consecutive positions along the sensestrand nucleotide sequence). Alternatively, nucleotides of the sensestrand that form mismatched base pairs with the antisense strandsequence can be interspersed by nucleotides that base pair with theantisense strand sequence (e.g., for a DsiRNAmm possessing mismatchednucleotides at positions 3 and 6, but not at positions 4 and 5, themismatched residues of sense strand positions 3 and 6 are interspersedby two nucleotides that form matched base pairs with correspondingresidues of the antisense strand). For example, two residues of thesense strand (located within the mismatch-tolerant region of the sensestrand) that form mismatched base pairs with the corresponding antisensestrand sequence can occur with zero, one, two, three, four or fivematched base pairs located between these mismatched base pairs.

For certain DsiRNAmm agents possessing three mismatched base pairs,mismatches can occur consecutively (e.g., in a triplet along the sensestrand nucleotide sequence). Alternatively, nucleotides of the sensestrand that form mismatched base pairs with the antisense strandsequence can be interspersed by nucleotides that form matched base pairswith the antisense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 3, 4 and 8, but not at positions 5,6 and 7, the mismatched residues of sense strand positions 3 and 4 areadjacent to one another, while the mismatched residues of sense strandpositions 4 and 8 are interspersed by three nucleotides that formmatched base pairs with corresponding residues of the antisense strand).For example, three residues of the sense strand (located within themismatch-tolerant region of the sense strand) that form mismatched basepairs with the corresponding antisense strand sequence can occur withzero, one, two, three or four matched base pairs located between any twoof these mismatched base pairs.

For certain DsiRNAmm agents possessing four mismatched base pairs,mismatches can occur consecutively (e.g., in a quadruplet along thesense strand nucleotide sequence). Alternatively, nucleotides of thesense strand that form mismatched base pairs with the antisense strandsequence can be interspersed by nucleotides that form matched base pairswith the antisense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 3, 5, 7 and 8, but not at positions4 and 6, the mismatched residues of sense strand positions 7 and 8 areadjacent to one another, while the mismatched residues of sense strandpositions 3 and 5 are interspersed by one nucleotide that forms amatched base pair with the corresponding residue of the antisensestrand—similarly, the the mismatched residues of sense strand positions5 and 7 are also interspersed by one nucleotide that forms a matchedbase pair with the corresponding residue of the antisense strand). Forexample, four residues of the sense strand (located within themismatch-tolerant region of the sense strand) that form mismatched basepairs with the corresponding antisense strand sequence can occur withzero, one, two or three matched base pairs located between any two ofthese mismatched base pairs.

In another embodiment, a DsiRNAmm of the invention comprises a mismatchtolerant region which possesses a single mismatched base pair nucleotideat any one of positions 13, 14, 15, 16, 17, 18, 19, 20 or 21 of theantisense strand of a left-hand extended DsiRNA (where position 1 is the5′ terminal nucleotide of the antisense strand and position 13 is thenucleotide residue of the antisense strand that is immediately 3′(downstream) in the antisense strand of the projected Ago2 cut site ofthe target RNA sequence sufficiently complementary to the antisensestrand sequence). In certain embodiments, for a DsiRNAmm that possessesa mismatched base pair nucleotide at any of positions 13, 14, 15, 16,17, 18, 19, 20 or 21 of the antisense strand with respect to the sensestrand of the DsiRNAmm, the mismatched base pair nucleotide of theantisense strand not only forms a mismatched base pair with the DsiRNAmmsense strand sequence, but also forms a mismatched base pair with aDsiRNAmm target RNA sequence (thus, complementarity between theantisense strand sequence and the sense strand sequence is disrupted atthe mismatched base pair within the DsiRNAmm, and complementarity issimilarly disrupted between the antisense strand sequence of theDsiRNAmm and the target RNA sequence). In alternative embodiments, themismatch base pair nucleotide of the antisense strand of a DsiRNAmm onlyforms a mismatched base pair with a corresponding nucleotide of thesense strand sequence of the DsiRNAmm, yet base pairs with itscorresponding target RNA sequence nucleotide (thus, complementaritybetween the antisense strand sequence and the sense strand sequence isdisrupted at the mismatched base pair within the DsiRNAmm, yetcomplementarity is maintained between the antisense strand sequence ofthe DsiRNAmm and the target RNA sequence).

A DsiRNAmm of the invention that possesses a single mismatched base pairwithin the mismatch-tolerant region as described above (e.g., a DsiRNAmmharboring a mismatched nucleotide residue at positions 13, 14, 15, 16,17, 18, 19, 20 or 21 of the antisense strand) can further include one,two or even three additional mismatched base pairs. In preferredembodiments, these one, two or three additional mismatched base pairs ofthe DsiRNAmm occur at position(s) 13, 14, 15, 16, 17, 18, 19, 20 and/or21 of the antisense strand (and at corresponding residues of the sensestrand). In one embodiment where one additional mismatched base pair ispresent within a DsiRNAmm, the two mismatched base pairs of theantisense strand can occur, e.g., at nucleotides of both position 14 andposition 18 of the antisense strand (with mismatch also occurring atcorresponding nucleotide residues of the sense strand).

In DsiRNAmm agents possessing two mismatched base pairs, mismatches canoccur consecutively (e.g., at consecutive positions along the antisensestrand nucleotide sequence). Alternatively, nucleotides of the antisensestrand that form mismatched base pairs with the sense strand sequencecan be interspersed by nucleotides that base pair with the sense strandsequence (e.g., for a DsiRNAmm possessing mismatched nucleotides atpositions 13 and 16, but not at positions 14 and 15, the mismatchedresidues of antisense strand positions 13 and 16 are interspersed by twonucleotides that form matched base pairs with corresponding residues ofthe sense strand). For example, two residues of the antisense strand(located within the mismatch-tolerant region of the sense strand) thatform mismatched base pairs with the corresponding sense strand sequencecan occur with zero, one, two, three, four, five, six or seven matchedbase pairs located between these mismatched base pairs.

For certain DsiRNAmm agents possessing three mismatched base pairs,mismatches can occur consecutively (e.g., in a triplet along theantisense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the sense strandsequence can be interspersed by nucleotides that form matched base pairswith the sense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 13, 14 and 18, but not at positions15, 16 and 17, the mismatched residues of antisense strand positions 13and 14 are adjacent to one another, while the mismatched residues ofantisense strand positions 14 and 18 are interspersed by threenucleotides that form matched base pairs with corresponding residues ofthe sense strand). For example, three residues of the antisense strand(located within the mismatch-tolerant region of the antisense strand)that form mismatched base pairs with the corresponding sense strandsequence can occur with zero, one, two, three, four, five or six matchedbase pairs located between any two of these mismatched base pairs.

For certain DsiRNAmm agents possessing four mismatched base pairs,mismatches can occur consecutively (e.g., in a quadruplet along theantisense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the sense strandsequence can be interspersed by nucleotides that form matched base pairswith the sense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 13, 15, 17 and 18, but not atpositions 14 and 16, the mismatched residues of antisense strandpositions 17 and 18 are adjacent to one another, while the mismatchedresidues of antisense strand positions 13 and 15 are interspersed by onenucleotide that forms a matched base pair with the corresponding residueof the sense strand—similarly, the the mismatched residues of antisensestrand positions 15 and 17 are also interspersed by one nucleotide thatforms a matched base pair with the corresponding residue of the sensestrand). For example, four residues of the antisense strand (locatedwithin the mismatch-tolerant region of the antisense strand) that formmismatched base pairs with the corresponding sense strand sequence canoccur with zero, one, two, three, four or five matched base pairslocated between any two of these mismatched base pairs.

In a further embodiment, a DsiRNAmm of the invention possesses a singlemismatched base pair nucleotide at any one of positions 11, 12, 13, 14,15, 16, 17, 18 or 19 of the antisense strand of a left-hand extendedDsiRNA (where position 1 is the 5′ terminal nucleotide of the antisensestrand and position 11 is the nucleotide residue of the antisense strandthat is immediately 3′ (downstream) in the antisense strand of theprojected Ago2 cut site of the target RNA sequence sufficientlycomplementary to the antisense strand sequence). In certain embodiments,for a DsiRNAmm that possesses a mismatched base pair nucleotide at anyof positions 11, 12, 13, 14, 15, 16, 17, 18 or 19 of the antisensestrand with respect to the sense strand of the DsiRNAmm, the mismatchedbase pair nucleotide of the antisense strand not only forms a mismatchedbase pair with the DsiRNAmm sense strand sequence, but also forms amismatched base pair with a DsiRNAmm target RNA sequence (thus,complementarity between the antisense strand sequence and the sensestrand sequence is disrupted at the mismatched base pair within theDsiRNAmm, and complementarity is similarly disrupted between theantisense strand sequence of the DsiRNAmm and the target RNA sequence).In alternative embodiments, the mismatch base pair nucleotide of theantisense strand of a DsiRNAmm only forms a mismatched base pair with acorresponding nucleotide of the sense strand sequence of the DsiRNAmm,yet this same antisense strand nucleotide base pairs with itscorresponding target RNA sequence nucleotide (thus, complementaritybetween the antisense strand sequence and the sense strand sequence isdisrupted at the mismatched base pair within the DsiRNAmm, yetcomplementarity is maintained between the antisense strand sequence ofthe DsiRNAmm and the target RNA sequence).

A DsiRNAmm of the invention that possesses a single mismatched base pairwithin the mismatch-tolerant region as described above (e.g., a DsiRNAmmharboring a mismatched nucleotide residue at positions 11, 12, 13, 14,15, 16, 17, 18 or 19 of the antisense strand) can further include one,two or even three additional mismatched base pairs. In preferredembodiments, these one, two or three additional mismatched base pairs ofthe DsiRNAmm occur at position(s) 11, 12, 13, 14, 15, 16, 17, 18 and/or19 of the antisense strand (and at corresponding residues of the sensestrand). In one embodiment where one additional mismatched base pair ispresent within a DsiRNAmm, the two mismatched base pairs of theantisense strand can occur, e.g., at nucleotides of both position 14 andposition 18 of the antisense strand (with mismatch also occurring atcorresponding nucleotide residues of the sense strand).

In DsiRNAmm agents possessing two mismatched base pairs, mismatches canoccur consecutively (e.g., at consecutive positions along the antisensestrand nucleotide sequence). Alternatively, nucleotides of the antisensestrand that form mismatched base pairs with the sense strand sequencecan be interspersed by nucleotides that base pair with the sense strandsequence (e.g., for a DsiRNAmm possessing mismatched nucleotides atpositions 12 and 15, but not at positions 13 and 14, the mismatchedresidues of antisense strand positions 12 and 15 are interspersed by twonucleotides that form matched base pairs with corresponding residues ofthe sense strand). For example, two residues of the antisense strand(located within the mismatch-tolerant region of the sense strand) thatform mismatched base pairs with the corresponding sense strand sequencecan occur with zero, one, two, three, four, five, six or seven matchedbase pairs located between these mismatched base pairs.

For certain DsiRNAmm agents possessing three mismatched base pairs,mismatches can occur consecutively (e.g., in a triplet along theantisense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the sense strandsequence can be interspersed by nucleotides that form matched base pairswith the sense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 13, 14 and 18, but not at positions15, 16 and 17, the mismatched residues of antisense strand positions 13and 14 are adjacent to one another, while the mismatched residues ofantisense strand positions 14 and 18 are interspersed by threenucleotides that form matched base pairs with corresponding residues ofthe sense strand). For example, three residues of the antisense strand(located within the mismatch-tolerant region of the antisense strand)that form mismatched base pairs with the corresponding sense strandsequence can occur with zero, one, two, three, four, five or six matchedbase pairs located between any two of these mismatched base pairs.

For certain DsiRNAmm agents possessing four mismatched base pairs,mismatches can occur consecutively (e.g., in a quadruplet along theantisense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the sense strandsequence can be interspersed by nucleotides that form matched base pairswith the sense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 13, 15, 17 and 18, but not atpositions 14 and 16, the mismatched residues of antisense strandpositions 17 and 18 are adjacent to one another, while the mismatchedresidues of antisense strand positions 13 and 15 are interspersed by onenucleotide that forms a matched base pair with the corresponding residueof the sense strand—similarly, the the mismatched residues of antisensestrand positions 15 and 17 are also interspersed by one nucleotide thatforms a matched base pair with the corresponding residue of the sensestrand). For example, four residues of the antisense strand (locatedwithin the mismatch-tolerant region of the antisense strand) that formmismatched base pairs with the corresponding sense strand sequence canoccur with zero, one, two, three, four or five matched base pairslocated between any two of these mismatched base pairs.

In an additional embodiment, a DsiRNAmm of the invention possesses asingle mismatched base pair nucleotide at any one of positions 15, 16,17, 18, 19, 20, 21, 22 or 23 of the antisense strand of a left-handextended DsiRNA (where position 1 is the 5′ terminal nucleotide of theantisense strand and position 15 is the nucleotide residue of theantisense strand that is immediately 3′ (downstream) in the antisensestrand of the projected Ago2 cut site of the target RNA sequencesufficiently complementary to the antisense strand sequence). In certainembodiments, for a DsiRNAmm that possesses a mismatched base pairnucleotide at any of positions 15, 16, 17, 18, 19, 20, 21, 22 or 23 ofthe antisense strand with respect to the sense strand of the DsiRNAmm,the mismatched base pair nucleotide of the antisense strand not onlyforms a mismatched base pair with the DsiRNAmm sense strand sequence,but also forms a mismatched base pair with a DsiRNAmm target RNAsequence (thus, complementarity between the antisense strand sequenceand the sense strand sequence is disrupted at the mismatched base pairwithin the DsiRNAmm, and complementarity is similarly disrupted betweenthe antisense strand sequence of the DsiRNAmm and the target RNAsequence). In alternative embodiments, the mismatch base pair nucleotideof the antisense strand of a DsiRNAmm only forms a mismatched base pairwith a corresponding nucleotide of the sense strand sequence of theDsiRNAmm, yet this same antisense strand nucleotide base pairs with itscorresponding target RNA sequence nucleotide (thus, complementaritybetween the antisense strand sequence and the sense strand sequence isdisrupted at the mismatched base pair within the DsiRNAmm, yetcomplementarity is maintained between the antisense strand sequence ofthe DsiRNAmm and the target RNA sequence).

A DsiRNAmm of the invention that possesses a single mismatched base pairwithin the mismatch-tolerant region as described above (e.g., a DsiRNAmmharboring a mismatched nucleotide residue at positions 15, 16, 17, 18,19, 20, 21, 22 or 23 of the antisense strand) can further include one,two or even three additional mismatched base pairs. In preferredembodiments, these one, two or three additional mismatched base pairs ofthe DsiRNAmm occur at position(s) 15, 16, 17, 18, 19, 20, 21, 22 and/or23 of the antisense strand (and at corresponding residues of the sensestrand). In one embodiment where one additional mismatched base pair ispresent within a DsiRNAmm, the two mismatched base pairs of theantisense strand can occur, e.g., at nucleotides of both position 16 andposition 20 of the antisense strand (with mismatch also occurring atcorresponding nucleotide residues of the sense strand).

In DsiRNAmm agents possessing two mismatched base pairs, mismatches canoccur consecutively (e.g., at consecutive positions along the antisensestrand nucleotide sequence). Alternatively, nucleotides of the antisensestrand that form mismatched base pairs with the sense strand sequencecan be interspersed by nucleotides that base pair with the sense strandsequence (e.g., for a DsiRNAmm possessing mismatched nucleotides atpositions 16 and 20, but not at positions 17, 18 and 19, the mismatchedresidues of antisense strand positions 16 and 20 are interspersed bythree nucleotides that form matched base pairs with correspondingresidues of the sense strand). For example, two residues of theantisense strand (located within the mismatch-tolerant region of thesense strand) that form mismatched base pairs with the correspondingsense strand sequence can occur with zero, one, two, three, four, five,six or seven matched base pairs located between these mismatched basepairs.

For certain DsiRNAmm agents possessing three mismatched base pairs,mismatches can occur consecutively (e.g., in a triplet along theantisense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the sense strandsequence can be interspersed by nucleotides that form matched base pairswith the sense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 16, 17 and 21, but not at positions18, 19 and 20, the mismatched residues of antisense strand positions 16and 17 are adjacent to one another, while the mismatched residues ofantisense strand positions 17 and 21 are interspersed by threenucleotides that form matched base pairs with corresponding residues ofthe sense strand). For example, three residues of the antisense strand(located within the mismatch-tolerant region of the antisense strand)that form mismatched base pairs with the corresponding sense strandsequence can occur with zero, one, two, three, four, five or six matchedbase pairs located between any two of these mismatched base pairs.

For certain DsiRNAmm agents possessing four mismatched base pairs,mismatches can occur consecutively (e.g., in a quadruplet along theantisense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the sense strandsequence can be interspersed by nucleotides that form matched base pairswith the sense strand sequence (e.g., for a DsiRNAmm possessingmismatched nucleotides at positions 17, 19, 21 and 22, but not atpositions 18 and 20, the mismatched residues of antisense strandpositions 21 and 22 are adjacent to one another, while the mismatchedresidues of antisense strand positions 17 and 19 are interspersed by onenucleotide that forms a matched base pair with the corresponding residueof the sense strand—similarly, the the mismatched residues of antisensestrand positions 19 and 21 are also interspersed by one nucleotide thatforms a matched base pair with the corresponding residue of the sensestrand). For example, four residues of the antisense strand (locatedwithin the mismatch-tolerant region of the antisense strand) that formmismatched base pairs with the corresponding sense strand sequence canoccur with zero, one, two, three, four or five matched base pairslocated between any two of these mismatched base pairs.

For reasons of clarity, the location(s) of mismatched nucleotideresidues within the above DsiRNAmm agents are numbered in reference tothe 5′ terminal residue of either sense or antisense strands of theDsiRNAmm. The numbering of positions located within themismatch-tolerant region (mismatch region) of the antisense strand canshift with variations in the proximity of the 5′ terminus of theantisense strand to the projected Ago2 cleavage site. Thus, thelocation(s) of preferred mismatch sites within either antisense strandor sense strand can also be identified as the permissible proximity ofsuch mismatches to the projected Ago2 cut site. Accordingly, in onepreferred embodiment, the position of a mismatch nucleotide of the sensestrand of a DsiRNAmm is the nucleotide residue of the sense strand thatis located immediately 5′ (upstream) of the projected Ago2 cleavage siteof the corresponding target RNA sequence. In other preferredembodiments, a mismatch nucleotide of the sense strand of a DsiRNAmm ispositioned at the nucleotide residue of the sense strand that is locatedtwo nucleotides 5′ (upstream) of the projected Ago2 cleavage site, threenucleotides 5′ (upstream) of the projected Ago2 cleavage site, fournucleotides 5′ (upstream) of the projected Ago2 cleavage site, fivenucleotides 5′ (upstream) of the projected Ago2 cleavage site, sixnucleotides 5′ (upstream) of the projected Ago2 cleavage site, sevennucleotides 5′ (upstream) of the projected Ago2 cleavage site, eightnucleotides 5′ (upstream) of the projected Ago2 cleavage site, or ninenucleotides 5′ (upstream) of the projected Ago2 cleavage site.

Exemplary single mismatch-containing, 5′guide single strand extendedDsiRNAs (DsiRNAmm) include the following structures (suchmismatch-containing structures may also be incorporated into otherexemplary DsiRNA structures shown herein).

5′-XX^(M)XXXXXXXXXXXXXXXXXXXX_(N*)DD-3′3′-YXX_(M)XXXXXXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′5′-XXX^(M)XXXXXXXXXXXXXXXXXXX_(N*)DD-3′3′-YXXX_(M)XXXXXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′5′-XXXX^(M)XXXXXXXXXXXXXXXXXX_(N*)DD-3′3′-YXXXX_(M)XXXXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′5′-XXXXX^(M)XXXXXXXXXXXXXXXXX_(N*)DD-3′3′-YXXXXX_(M)XXXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′5′-XXXXXX^(M)XXXXXXXXXXXXXXXX_(N*)DD-3′3′-YXXXXXX_(M)XXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′5′-XXXXXXX^(M)XXXXXXXXXXXXXXX_(N*)DD-3′3′-YXXXXXXX_(M)XXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′5′-XXXXXXXX^(M)XXXXXXXXXXXXXX_(N*)DD-3′3′-YXXXXXXXX_(M)XXXXXXXXXXXXXX_(N*)XXZ_(N)-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “Z”=DNA, RNA, or modifiednucleotide, “N”=1 to 50 or more, but is optionally 1-30 or, optionally1-15 or, optionally, 1-10. “N*”=0 to 15 or more, but is optionally 0, 1,2, 3, 4, or 5, and “D”=DNA, “M”=Nucleic acid residues (RNA, DNA ornon-natural or modified nucleic acids) that do not base pair (hydrogenbond) with corresponding “M” residues of otherwise complementary strandwhen strands are annealed. Any of the residues of such agents canoptionally be 2′-O-methyl RNA monomers—alternating positioning of2′-O-methyl RNA monomers that commences from the 3′-terminal residue ofthe bottom (second) strand, as shown above, can also be used in theabove DsiRNAmm agents. For the above mismatch structures, the top strandis the sense strand, and the bottom strand is the antisense strand.

In certain embodiments, a DsiRNA of the invention can contain mismatchesthat exist in reference to the target RNA sequence yet do notnecessarily exist as mismatched base pairs within the two strands of theDsiRNA—thus, a DsiRNA can possess perfect complementarity between firstand second strands of a DsiRNA, yet still possess mismatched residues inreference to a target RNA (which, in certain embodiments, may beadvantageous in promoting efficacy and/or potency and/or duration ofeffect). In certain embodiments, where mismatches occur betweenantisense strand and target RNA sequence, the position of a mismatch islocated within the antisense strand at a position(s) that corresponds toa sequence of the sense strand located 5′ of the projected Ago2 cut siteof the target region—e.g., antisense strand residue(s) positioned withinthe antisense strand to the 3′ of the antisense residue which iscomplementary to the projected Ago2 cut site of the target sequence.

Exemplary 25/27mer DsiRNAs that harbor a single mismatched residue inreference to target sequences include the following preferredstructures.

Target RNA Sequence: 5′-. . AXXXXXXXXXXXXXXXXXXXX . . .-3′DsiRNAmm Sense Strand: 5′-XXXXXXXXXXXXXXXXXXXXXXX_(N*)DD-3′DsiRNAmm Antisense Strand: 3′-EXXXXXXXXXXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′Target RNA Sequence: 5′-. . XAXXXXXXXXXXXXXXXXXXX . . .-3′DsiRNAmm Sense Strand: 5′-XXXXXXXXXXXXXXXXXXXXXXX_(N*)DD-3′DsiRNAmm Antisense Strand: 3′-XEXXXXXXXXXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′Target RNA Sequence: 5′-. . . AXXXXXXXXXXXXXXXXXX . . .-3′DsiRNAmm Sense Strand: 5′-BXXXXXXXXXXXXXXXXXXXXXX_(N*)DD-3′DsiRNAmm Antisense Strand: 3′-XXEXXXXXXXXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′Target RNA Sequence: 5′-. . . XAXXXXXXXXXXXXXXXXX . . .-3′DsiRNAmm Sense Strand: 5′-XBXXXXXXXXXXXXXXXXXXXXX_(N*)DD-3′DsiRNAmm Antisense Strand: 3′-XXXEXXXXXXXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′Target RNA Sequence: 5′-. . . XXAXXXXXXXXXXXXXXXX . . .-3′DsiRNAmm Sense Strand: 5′-XXBXXXXXXXXXXXXXXXXXXXX_(N*)DD-3′DsiRNAmm Antisense Strand: 3′-XXXXEXXXXXXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′Target RNA Sequence: 5′-. . . XXXAXXXXXXXXXXXXXXX . . .-3′DsiRNAmm Sense Strand: 5′-XXXBXXXXXXXXXXXXXXXXXXX_(N*)DD-3′DsiRNAmm Antisense Strand: 3′-XXXXXEXXXXXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′Target RNA Sequence: 5′-. . . XXXXAXXXXXXXXXXXXXX . . .-3′DsiRNAmm Sense Strand: 5′-XXXXBXXXXXXXXXXXXXXXXXX_(N*)DD-3′DsiRNAmm Antisense Strand: 3′-XXXXXXEXXXXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′Target RNA Sequence: 5′-. . . XXXXXAXXXXXXXXXXXXX . . .-3′DsiRNAmm Sense Strand: 5′-XXXXXBXXXXXXXXXXXXXXXXX_(N*)DD-3′DsiRNAmm Antisense Strand: 3′-XXXXXXXEXXXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′Target RNA Sequence: 5′-. . . XXXXXXAXXXXXXXXXXXX . . .-3′DsiRNAmm Sense Strand: 5′-XXXXXXBXXXXXXXXXXXXXXXX_(N*)DD-3′DsiRNAmm Antisense Strand: 3′-XXXXXXXXEXXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′Target RNA Sequence: 5′-. . . XXXXXXXAXXXXXXXXXXX . . .-3′DsiRNAmm Sense Strand: 5′-XXXXXXXBXXXXXXXXXXXXXXX_(N*)DD-3′DsiRNAmm Antisense Strand: 3′-XXXXXXXXXEXXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′Target RNA Sequence: 5′-. . . XXXXXXXXAXXXXXXXXXX . . .-3′DsiRNAmm Sense Strand: 5′-XXXXXXXXBXXXXXXXXXXXXXX_(N*)DD-3′DsiRNAmm Antisense Strand: 3′-XXXXXXXXXXEXXXXXXXXXXXXXX_(N*)XXZ_(N)-5′wherein “X”=RNA, “Y” is an optional overhang domain comprised of 0-10RNA monomers that are optionally 2′-O-methyl RNA monomers—in certainembodiments, “Y” is an overhang domain comprised of 1-4 RNA monomersthat are optionally 2′-O-methyl RNA monomers, “Z”=DNA, RNA, or modifiednucleotide, “N”=1 to 50 or more, but is optionally 1-30 or, optionally1-15 or, optionally, 1-10. “N*”=0 to 15 or more, but is optionally 0, 1,2, 3, 4, or 5, “D”=DNA, “p”=a phosphate group, “E”=Nucleic acid residues(RNA, DNA or non-natural or modified nucleic acids) that do not basepair (hydrogen bond) with corresponding “A” RNA residues of otherwisecomplementary (target) strand when strands are annealed, yet optionallydo base pair with corresponding “B” residues (“B” residues are also RNA,DNA or non-natural or modified nucleic acids).Any of the residues of such agents can optionally be 2′-O-methyl RNAmonomers—e.g., alternating positioning of 2′-O-methyl RNA monomers thatcommences from the 3′-terminal residue of the bottom (second) strand, asshown above, or other patterns of 2′-O-methyl and/or other modificationsas described herein can also be used in the above DsiRNA agents.

In addition to the above-exemplified structures, DsiRNAs of theinvention can also possess one, two or three additional residues thatform further mismatches with the target RNA sequence. Such mismatchescan be consecutive, or can be interspersed by nucleotides that formmatched base pairs with the target RNA sequence. Where interspersed bynucleotides that form matched base pairs, mismatched residues can bespaced apart from each other within a single strand at an interval ofone, two, three, four, five, six, seven or even eight base pairednucleotides between such mismatch-forming residues.

As for the above-described DsiRNAmm agents, a preferred location withinDsiRNAs for antisense strand nucleotides that form mismatched base pairswith target RNA sequence (yet may or may not form mismatches withcorresponding sense strand nucleotides) is within the antisense strandregion that is located 3′ (downstream) of the antisense strand sequencewhich is complementary to the projected Ago2 cut site of the DsiRNA.Thus, in one preferred embodiment, the position of a mismatch nucleotide(in relation to the target RNA sequence) of the antisense strand of aDsiRNAmm is the nucleotide residue of the antisense strand that islocated immediately 3′ (downstream) within the antisense strand sequenceof the projected Ago2 cleavage site of the corresponding target RNAsequence. In other preferred embodiments, a mismatch nucleotide of theantisense strand of a DsiRNAmm (in relation to the target RNA sequence)is positioned at the nucleotide residue of the antisense strand that islocated two nucleotides 3′ (downstream) of the corresponding projectedAgo2 cleavage site, three nucleotides 3′ (downstream) of thecorresponding projected Ago2 cleavage site, four nucleotides 3′(downstream) of the corresponding projected Ago2 cleavage site, fivenucleotides 3′ (downstream) of the corresponding projected Ago2 cleavagesite, six nucleotides 3′ (downstream) of the projected Ago2 cleavagesite, seven nucleotides 3′ (downstream) of the projected Ago2 cleavagesite, eight nucleotides 3′ (downstream) of the projected Ago2 cleavagesite, or nine nucleotides 3′ (downstream) of the projected Ago2 cleavagesite.

In DsiRNA agents possessing two mismatch-forming nucleotides of theantisense strand (where mismatch-forming nucleotides are mismatchforming in relation to target RNA sequence), mismatches can occurconsecutively (e.g., at consecutive positions along the antisense strandnucleotide sequence). Alternatively, nucleotides of the antisense strandthat form mismatched base pairs with the target RNA sequence can beinterspersed by nucleotides that base pair with the target RNA sequence(e.g., for a DsiRNA possessing mismatch-forming nucleotides at positions13 and 16 (starting from the 5′ terminus (position 1) of the antisensestrand), but not at positions 14 and 15, the mismatched residues ofsense strand positions 13 and 16 are interspersed by two nucleotidesthat form matched base pairs with corresponding residues of the targetRNA sequence). For example, two residues of the antisense strand(located within the mismatch-tolerant region of the antisense strand)that form mismatched base pairs with the corresponding target RNAsequence can occur with zero, one, two, three, four or five matched basepairs (with respect to target RNA sequence) located between thesemismatch-forming base pairs.

For certain DsiRNAs possessing three mismatch-forming base pairs(mismatch-forming with respect to target RNA sequence), mismatch-formingnucleotides can occur consecutively (e.g., in a triplet along theantisense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the target RNAsequence can be interspersed by nucleotides that form matched base pairswith the target RNA sequence (e.g., for a DsiRNA possessing mismatchednucleotides at positions 13, 14 and 18, but not at positions 15, 16 and17, the mismatch-forming residues of antisense strand positions 13 and14 are adjacent to one another, while the mismatch-forming residues ofantisense strand positions 14 and 18 are interspersed by threenucleotides that form matched base pairs with corresponding residues ofthe target RNA). For example, three residues of the antisense strand(located within the mismatch-tolerant region of the antisense strand)that form mismatched base pairs with the corresponding target RNAsequence can occur with zero, one, two, three or four matched base pairslocated between any two of these mismatch-forming base pairs.

For certain DsiRNAs possessing four mismatch-forming base pairs(mismatch-forming with respect to target RNA sequence), mismatch-formingnucleotides can occur consecutively (e.g., in a quadruplet along thesense strand nucleotide sequence). Alternatively, nucleotides of theantisense strand that form mismatched base pairs with the target RNAsequence can be interspersed by nucleotides that form matched base pairswith the target RNA sequence (e.g., for a DsiRNA possessingmismatch-forming nucleotides at positions 13, 15, 17 and 18, but not atpositions 14 and 16, the mismatch-forming residues of antisense strandpositions 17 and 18 are adjacent to one another, while themismatch-forming residues of antisense strand positions 13 and 15 areinterspersed by one nucleotide that forms a matched base pair with thecorresponding residue of the target RNA sequence—similarly, themismatch-forming residues of antisense strand positions 15 and 17 arealso interspersed by one nucleotide that forms a matched base pair withthe corresponding residue of the target RNA sequence). For example, fourresidues of the antisense strand (located within the mismatch-tolerantregion of the antisense strand) that form mismatched base pairs with thecorresponding target RNA sequence can occur with zero, one, two or threematched base pairs located between any two of these mismatch-formingbase pairs.

The above DsiRNAmm and other DsiRNA structures are described in order toexemplify certain structures of DsiRNAmm and DsiRNA agents. Design ofthe above DsiRNAmm and DsiRNA structures can be adapted to generate,e.g., DsiRNAmm forms of a extended DsiRNA agent shown infra (including,e.g., design of mismatch-containing DsiRNAmm agents). As exemplifiedabove, DsiRNAs can also be designed that possess single mismatches (ortwo, three or four mismatches) between the antisense strand of theDsiRNA and a target sequence, yet optionally can retain perfectcomplementarity between sense and antisense strand sequences of aDsiRNA.

It is further noted that the DsiRNA agents exemplified infra can alsopossess insertion/deletion (in/del) structures within theirdouble-stranded and/or target RNA-aligned structures. Accordingly, theDsiRNAs of the invention can be designed to possess in/del variationsin, e.g., antisense strand sequence as compared to target RNA sequenceand/or antisense strand sequence as compared to sense strand sequence,with preferred location(s) for placement of such in/del nucleotidescorresponding to those locations described above for positioning ofmismatched and/or mismatch-forming base pairs.

In certain embodiments, the “D” residues of any of the above structuresinclude at least one PS-DNA or PS-RNA. Optionally, the “D” residues ofany of the above structures include at least one modified nucleotidethat inhibits Dicer cleavage.

In one embodiment, the DsiRNA agent has an asymmetric structure, withthe sense strand having a 25-base pair length, the antisense strandhaving a 42-nucleotide length with a 2 base 3′-overhang (and, therefore,the DsiRNA agent possesses a 5′ overhang 15 nucleotides in length at the3′ end of the sense strand/5′ end of the antisense strand), and withdeoxyribonucleotides located at positions 24 and 25 of the sense strand(numbering from position 1 at the 5′ of the sense strand) and each basepaired with a cognate nucleotide of the antisense strand. The 5′overhang comprises a modified nucleotide, preferably a 2′-O-methylribonucleotide, and/or a phosphate backbone modification, preferablyphosphorothioate.

In another embodiment, the DsiRNA agent has a structure, with the sensestrand having a 40-nucleotide length, the antisense strand having a27-nucleotide length with a 2 base 3′-overhang (and, therefore, theDsiRNA agent possesses a 3′ overhang 15 nucleotides in length at the 3′end of the sense strand/5′ end of the antisense strand), and withdeoxyribonucleotides located at positions 24 and 25 of the sense strand(numbering from position 1 at the 5′ of the sense strand) and each basepaired with a cognate nucleotide of the antisense strand. The 3′overhang comprises a deoxyribonucleotide and/or a phosphate backbonemodification, preferably methylphosphonate.

Modification of DsiRNAs

One major factor that inhibits the effect of double stranded RNAs(“dsRNAs”) is the degradation of dsRNAs (e.g., siRNAs and DsiRNAs) bynucleases. A 3′-exonuclease is the primary nuclease activity present inserum and modification of the 3′-ends of antisense DNA oligonucleotidesis crucial to prevent degradation (Eder et al., 1991). An RNase-T familynuclease has been identified called ERI-1 which has 3′ to 5′ exonucleaseactivity that is involved in regulation and degradation of siRNAs(Kennedy et al., 2004; Hong et al., 2005). This gene is also known asThex1 (NM_02067) in mice or THEX1 (NM_153332) in humans and is involvedin degradation of histone mRNA; it also mediates degradation of3′-overhangs in siRNAs, but does not degrade duplex RNA (Yang et al.,2006). It is therefore reasonable to expect that 3′-end-stabilization ofdsRNAs, including the DsiRNAs of the instant invention, will improvestability.

XRN1 (NM_019001) is a 5′ to 3′ exonuclease that resides in P-bodies andhas been implicated in degradation of mRNA targeted by miRNA (Rehwinkelet al., 2005) and may also be responsible for completing degradationinitiated by internal cleavage as directed by a siRNA. XRN2 (NM_012255)is a distinct 5′ to 3′ exonuclease that is involved in nuclear RNAprocessing. Although not currently implicated in degradation orprocessing of siRNAs and miRNAs, these both are known nucleases that candegrade RNAs and may also be important to consider.

RNase A is a major endonuclease activity in mammals that degrades RNAs.It is specific for ssRNA and cleaves at the 3′-end of pyrimidine bases.SiRNA degradation products consistent with RNase A cleavage can bedetected by mass spectrometry after incubation in serum (Turner et al.,2007). The 3′-overhangs enhance the susceptibility of siRNAs to RNasedegradation. Depletion of RNase A from serum reduces degradation ofsiRNAs; this degradation does show some sequence preference and is worsefor sequences having poly A/U sequence on the ends (Haupenthal et al.,2006). This suggests the possibility that lower stability regions of theduplex may “breathe” and offer transient single-stranded speciesavailable for degradation by RNase A. RNase A inhibitors can be added toserum and improve siRNA longevity and potency (Haupenthal et al., 2007).

In 21mers, phosphorothioate or boranophosphate modifications directlystabilize the internucleoside phosphate linkage. Boranophosphatemodified RNAs are highly nuclease resistant, potent as silencing agents,and are relatively non-toxic. Boranophosphate modified RNAs cannot bemanufactured using standard chemical synthesis methods and instead aremade by in vitro transcription (IVT) (Hall et al., 2004 and Hall et al.,2006). Phosphorothioate (PS) modifications can be readily placed in anRNA duplex at any desired position and can be made using standardchemical synthesis methods, though the ability to use such modificationswithin an RNA duplex that retains RNA silencing activity can be limited.

In certain embodiments, the 5′ single strand extended region of theguide strand or 3′ single strand extended region of the passenger strandhas at least one phosphorothioate backbone modification. In someembodiments, every linkage of the 5′ single strand extended region ofthe guide strand or 3′ single strand extended region of the passengerstrand has a phosphorothioate backbone modification. In someembodiments, every linkage of the 5′ single strand extended region ofthe guide strand has a phosphorothioate backbone modification except thelinkage of the terminal 5′ nucleotide of the guide strand. In certainembodiments, the 5′ single strand extended region of the guide strand or3′ single strand extended region of the passenger strand has at leastone methylphosphonate backbone modification. In some embodiments, everylinkage of the 5′ single strand extended region of the guide strand or3′ single strand extended region of the passenger strand has amethylphosphonate backbone modification. In some embodiments, everylinkage of the 3′ single strand extended region of the passenger strandhas a phosphorothioate backbone modification except the terminal 5′nucleotide of the guide strand.

It is noted, however, that the PS modification shows dose-dependenttoxicity, so most investigators have recommended limited incorporationin siRNAs, historically favoring the 3′-ends where protection fromnucleases is most important (Harborth et al., 2003; Chiu and Rana, 2003;Braasch et al., 2003; Amarzguioui et al., 2003). More extensive PSmodification can be compatible with potent RNAi activity; however, useof sugar modifications (such as 2′-O-methyl RNA) may be superior (Chounget al., 2006).

A variety of substitutions can be placed at the 2′-position of theribose which generally increases duplex stability (T_(m)) and cangreatly improve nuclease resistance. 2′-O-methyl RNA is a naturallyoccurring modification found in mammalian ribosomal RNAs and transferRNAs. 2′-O-methyl modification in siRNAs is known, but the preciseposition of modified bases within the duplex is important to retainpotency and complete substitution of 2′-O-methyl RNA for RNA willinactivate the siRNA. For example, a pattern that employs alternating2′-O-methyl bases can have potency equivalent to unmodified RNA and isquite stable in serum (Choung et al., 2006; Czauderna et al., 2003).

The 2′-fluoro (2′-F) modification is also compatible with dsRNA (e.g.,siRNA and DsiRNA) function; it is most commonly placed at pyrimidinesites (due to reagent cost and availability) and can be combined with2′-O-methyl modification at purine positions; 2′-F purines are availableand can also be used. Heavily modified duplexes of this kind can bepotent triggers of RNAi in vitro (Allerson et al., 2005; Prakash et al.,2005; Kraynack and Baker, 2006) and can improve performance and extendduration of action when used in vivo (Morrissey et al., 2005a; Morrisseyet al., 2005b). A highly potent, nuclease stable, blunt 19mer duplexcontaining alternative 2′-F and 2′-O-Me bases is taught by Allerson. Inthis design, alternating 2′-O-Me residues are positioned in an identicalpattern to that employed by Czauderna, however the remaining RNAresidues are converted to 2′-F modified bases. A highly potent, nucleaseresistant siRNA employed by Morrissey employed a highly potent, nucleaseresistant siRNA in vivo. In addition to 2′-O-Me RNA and 2′-F RNA, thisduplex includes DNA, RNA, inverted abasic residues, and a 3′-terminal PSinternucleoside linkage. While extensive modification has certainbenefits, more limited modification of the duplex can also improve invivo performance and is both simpler and less costly to manufacture.Soutschek et al. (2004) employed a duplex in vivo and was mostly RNAwith two 2′-O-Me RNA bases and limited 3′-terminal PS internucleosidelinkages.

Locked nucleic acids (LNAs) are a different class of 2′-modificationthat can be used to stabilize dsRNA (e.g., siRNA and DsiRNA). Patternsof LNA incorporation that retain potency are more restricted than2′-O-methyl or 2′-F bases, so limited modification is preferred (Braaschet al., 2003; Grunweller et al., 2003; Elmen et al., 2005). Even withlimited incorporation, the use of LNA modifications can improve dsRNAperformance in vivo and may also alter or improve off target effectprofiles (Mook et al., 2007).

Synthetic nucleic acids introduced into cells or live animals can berecognized as “foreign” and trigger an immune response Immunestimulation constitutes a major class of off-target effects which candramatically change experimental results and even lead to cell death.The innate immune system includes a collection of receptor moleculesthat specifically interact with DNA and RNA that mediate theseresponses, some of which are located in the cytoplasm and some of whichreside in endosomes (Marques and Williams, 2005; Schlee et al., 2006).Delivery of siRNAs by cationic lipids or liposomes exposes the siRNA toboth cytoplasmic and endosomal compartments, maximizing the risk fortriggering a type 1 interferon (IFN) response both in vitro and in vivo(Morrissey et al., 2005b; Sioud and Sorensen, 2003; Sioud, 2005; Ma etal., 2005). RNAs transcribed within the cell are less immunogenic(Robbins et al., 2006) and synthetic RNAs that are immunogenic whendelivered using lipid-based methods can evade immune stimulation whenintroduced unto cells by mechanical means, even in vivo (Heidel et al.,2004). However, lipid based delivery methods are convenient, effective,and widely used. Some general strategy to prevent immune responses isneeded, especially for in vivo application where all cell types arepresent and the risk of generating an immune response is highest. Use ofchemically modified RNAs may solve most or even all of these problems.

Although certain sequence motifs are clearly more immunogenic thanothers, it appears that the receptors of the innate immune system ingeneral distinguish the presence or absence of certain basemodifications which are more commonly found in mammalian RNAs than inprokaryotic RNAs. For example, pseudouridine, N6-methyl-A, and2′-O-methyl modified bases are recognized as “self” and inclusion ofthese residues in a synthetic RNA can help evade immune detection(Kariko et al., 2005). Extensive 2′-modification of a sequence that isstrongly immunostimulatory as unmodified RNA can block an immuneresponse when administered to mice intravenously (Morrissey et al.,2005b). However, extensive modification is not needed to escape immunedetection and substitution of as few as two 2′-O-methyl bases in asingle strand of a siRNA duplex can be sufficient to block a type 1 IFNresponse both in vitro and in vivo; modified U and G bases are mosteffective (Judge et al., 2006). As an added benefit, selectiveincorporation of 2′-O-methyl bases can reduce the magnitude ofoff-target effects (Jackson et al., 2006). Use of 2′-O-methyl basesshould therefore be considered for all dsRNAs intended for in vivoapplications as a means of blocking immune responses and has the addedbenefit of improving nuclease stability and reducing the likelihood ofoff-target effects.

Although cell death can result from immune stimulation, assessing cellviability is not an adequate method to monitor induction of IFNresponses. IFN responses can be present without cell death, and celldeath can result from target knockdown in the absence of IFN triggering(for example, if the targeted gene is essential for cell viability).Relevant cytokines can be directly measured in culture medium and avariety of commercial kits exist which make performing such assaysroutine. While a large number of different immune effector molecules canbe measured, testing levels of IFN-α, TNF-α, and IL-6 at 4 and 24 hourspost transfection is usually sufficient for screening purposes. It isimportant to include a “transfection reagent only control” as cationiclipids can trigger immune responses in certain cells in the absence ofany nucleic acid cargo. Including controls for IFN pathway inductionshould be considered for cell culture work. It is essential to test forimmune stimulation whenever administering nucleic acids in vivo, wherethe risk of triggering IFN responses is highest.

Modifications can be included in the DsiRNA agents of the presentinvention so long as the modification does not prevent the DsiRNA agentfrom serving as a substrate for Dicer. Indeed, one surprising finding ofthe instant invention is that a 5′ extended single stranded nucleotideregion of the antisense strand or 3′ extended single stranded nucleotideregion of the sense strand can be attached to previously describedDsiRNA molecules, resulting in enhanced RNAi efficacy and duration,provided that such extension is performed in a region of the extendedmolecule that does not interfere with Dicer processing (e.g., 3′ of theDicer cleavage site of the sense strand/5′ of the Dicer cleavage site ofthe antisense strand). In one embodiment, one or more modifications aremade that enhance Dicer processing of the DsiRNA agent. In a secondembodiment, one or more modifications are made that result in moreeffective RNAi generation. In a third embodiment, one or moremodifications are made that support a greater RNAi effect. In a fourthembodiment, one or more modifications are made that result in greaterpotency per each DsiRNA agent molecule to be delivered to the cell.Modifications can be incorporated in the 3′-terminal region, the5′-terminal region, in both the 3′-terminal and 5′-terminal region or insome instances in various positions within the sequence. With therestrictions noted above in mind, any number and combination ofmodifications can be incorporated into the DsiRNA agent. Where multiplemodifications are present, they may be the same or different.Modifications to bases, sugar moieties, the phosphate backbone, andtheir combinations are contemplated. Either 5′-terminus can bephosphorylated.

Examples of modifications contemplated for the phosphate backboneinclude phosphonates, including methylphosphonate, phosphorothioate, andphosphotriester modifications such as alkylphosphotriesters, lockednucleic acids (LNA), morpholino, bicyclic furanose analogs and the like.Examples of modifications contemplated for the sugar moiety include2′-alkyl pyrimidine, such as 2′-O-methyl, 2′-fluoro, amino, and deoxymodifications and the like (see, e.g., Amarzguioui et al., 2003).Examples of modifications contemplated for the base groups includeabasic sugars, 2-O-alkyl modified pyrimidines, 4-thiouracil,5-bromouracil, 5-iodouracil, and 5-(3-aminoallyl)-uracil and the like.Locked nucleic acids, or LNA's, could also be incorporated. Many othermodifications are known and can be used so long as the above criteriaare satisfied. Examples of modifications are also disclosed in U.S. Pat.Nos. 5,684,143, 5,858,988 and 6,291,438 and in U.S. published patentapplication No. 2004/0203145 A1. Other modifications are disclosed inHerdewijn (2000), Eckstein (2000), Rusckowski et al. (2000), Stein etal. (2001); Vorobjev et al. (2001).

One or more modifications contemplated can be incorporated into eitherstrand. The placement of the modifications in the DsiRNA agent cangreatly affect the characteristics of the DsiRNA agent, includingconferring greater potency and stability, reducing toxicity, enhanceDicer processing, and minimizing an immune response. In one embodiment,the antisense strand or the sense strand or both strands have one ormore 2′-O-methyl modified nucleotides. In another embodiment, theantisense strand contains 2′-O-methyl modified nucleotides. In anotherembodiment, the antisense stand contains a 3′ overhang that comprises2′-O-methyl modified nucleotides. The antisense strand could alsoinclude additional 2′-O-methyl modified nucleotides.

In certain embodiments, the 5′ single strand extended region of theguide strand, 3′ single strand extended region of the passenger strand,or 5′single strand extended region of the passenger strand has at leastone modified nucleotide, optionally a 2′-O-methyl ribonucleotide. Insome embodiments, every nucleotide of the 5′ single strand extendedregion of the guide strand or 3′ single strand extended region of thepassenger strand is a modified ribonucleotide, optionally a 2′-O-methylribonucleotide. In certain embodiments, an oligonucleotide complementaryto the 5′ single strand extended region of the guide strand has at leastone modified nucleotide, optionally a 2′-O-methyl ribonucleotide. Insome embodiments, every nucleotide of an oligonucleotide complementaryto the 5′ single strand extended region of the guide strand is amodified nucleotide, optionally a 2′-O-methyl ribonucleotide.

In certain embodiments of the present invention, the DsiRNA agent hasone or more properties which enhance its processing by Dicer. Accordingto these embodiments, the DsiRNA agent has a length sufficient such thatit is processed by Dicer to produce an active siRNA and at least one ofthe following properties: (i) the DsiRNA agent is asymmetric, e.g., hasa 3′ overhang on the antisense strand and (ii) the DsiRNA agent has amodified 3′ end on the sense strand to direct orientation of Dicerbinding and processing of the dsRNA region to an active siRNA. Incertain such embodiments, the presence of one or more base paireddeoxyribonucleotides in a region of the sense strand that is 3′ to theprojected site of Dicer enzyme cleavage and corresponding region of theantisense strand that is 5′ of the projected site of Dicer enzymecleavage can also serve to orient such a molecule for appropriatedirectionality of Dicer enzyme cleavage.

In certain embodiments, the length of the 5′ single stranded antisenseextended region (5′ antisense extension) or 3′ single stranded senseextended region (3′ sense extension) is 1-30 nucleotides, optionally1-15 nucleotides, preferably 10-15 nucleotides, more preferably 11-15nucleotides. Thus, a single stranded extended DsiRNA of the instantinvention may possess a single strand extended region at the 5′ terminusof a antisense/guide strand or at the 3′ terminus of a sense/passengerstrand that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more (e.g.,31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more) nucleotides in length.

In some embodiments, the longest strand in the double stranded nucleicacid comprises 36-66 nucleotides. In one embodiment, the DsiRNA agenthas a structure such that the 5′ end of the antisense strand overhangsthe 3′ end of the sense strand, the 3′ end of the antisense strandoverhangs the 5′ end of the sense strand. In certain embodiments, the 5′overhang of the antisense strand is 1-30 nucleotides, and optionally is10-30 nucleotides, for example 15 nucleotides. In another embodiment,the DsiRNA agent has a structure such that the 3′ end of the sensestrand overhangs the 5′ end of the antisense strand, and the 3′ end ofthe antisense strand overhangs the 5′ end of the sense strand. Incertain embodiments, the 3′ overhang of the sense strand is 1-30nucleotides, and optionally is 10-30 nucleotides, for example 15nucleotides. In certain embodiments, the 3′ overhang of the antisensestrand is 1-10 nucleotides, and optionally is 1-6 nucleotides,preferably 1-4 nucleotides, for example 2 nucleotides. In anotherembodiment, the DsiRNA agent has a structure such that the 5′ end of thesense strand overhangs the 3′ end of the antisense strand. In certainembodiments, the 5′ overhang of the sense strand is 4-30 nucleotides,and optionally is 10-30 nucleotides, for example 15 nucleotides. Boththe sense and the antisense strand may also have a 5′ phosphate.

In certain embodiments, the sense strand of a DsiRNA of the inventionhas a total length of between 25 nucleotides and 30 or more nucleotides(e.g., the sense strand possesses a length of 25, 26, 27, 28, 29, 30 ormore (e.g., 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more)nucleotides). In certain embodiments, the length of the sense strand isbetween 25 nucleotides and 30 nucleotides, optionally between 26 and 30nucleotides, or, optionally, between 27 and 30 nucleotides in length. Inrelated embodiments, the antisense strand has a length of between 36 and66 or more nucleotides (e.g., the sense strand possesses a length of236, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 or more (e.g., 67,28, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 or more)nucleotides). In certain such embodiments, the antisense strand has alength of between 37 and 57 nucleotides in length, or between 37 and 52nucleotides in length, or between 37 and 47 nucleotides in length, orbetween 42 and 62 nucleotides in length, or between 42 and 57nucleotides in length, or between 42 and 47 nucleotides in length.

In certain embodiments, the sense strand of a DsiRNA of the inventionhas a total length of between 25 nucleotides and 60 or more nucleotides(e.g., the sense strand possesses a length of 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more (e.g., 61, 62,63, 64, 65, 66, 67, 68, 69, 70 or more) nucleotides). In certainembodiments, the length of the sense strand is between 25 nucleotidesand 30 nucleotides, optionally between 35 and 55 nucleotides, or,optionally, between 40 and 55 nucleotides in length, or, optionally,between 40 and 60 nucleotides in length, or, optionally, between 45 and60 nucleotides in length. In related embodiments, the antisense strandhas a length of between 25 and 36 or more nucleotides (e.g., the sensestrand possesses a length of 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,or more (e.g., 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 ormore) nucleotides). In certain such embodiments, the antisense strandhas a length of between 27 and 32 nucleotides in length.

In certain embodiments, the presence of one or more base paireddeoxyribonucleotides in a region of the sense strand that is 3′ of theprojected site of Dicer enzyme cleavage and corresponding region of theantisense strand that is 5′ of the projected site of Dicer enzymecleavage can serve to direct Dicer enzyme cleavage of such a molecule.While certain exemplified agents of the invention possess a sense stranddeoxyribonucleotide that is located at position 24 or more 3′ whencounting from position 1 at the 5′ end of the sense strand, and havingthis position 24 or more 3′ deoxyribonucleotide of the sense strand basepairing with a cognate deoxyribonucleotide of the antisense strand, insome embodiments, it is also possible to direct Dicer to cleave ashorter product, e.g., a 19mer or a 20mer via inclusion ofdeoxyribonucleotide residues at, e.g., position 20 of the sense strand.Such a position 20 deoxyribonucleotide base pairs with a correspondingdeoxyribonucleotide of the antisense strand, thereby directingDicer-mediated excision of a 19mer as the most prevalent Dicer product(it is noted that the antisense strand can also comprise one or twodeoxyribonucleotide residues immediately 3′ of the antisense residuethat base pairs with the position 20 deoxyribonucleotide residue of thesense strand in such embodiments, to further direct Dicer cleavage ofthe antisense strand). In such embodiments, the double-stranded DNAregion (which is inclusive of modified nucleic acids that block Dicercleavage) will generally possess a length of greater than 1 or 2 basepairs (e.g., 3 to 5 base pairs or more), in order to direct Dicercleavage to generate what is normally a non-preferred length of Dicercleavage product. A parallel approach can also be taken to direct Dicerexcision of 20mer siRNAs, with the positioning of the firstdeoxyribonucleotide residue of the sense strand (when surveying thesense strand from position 1 at the 5′ terminus of the sense strand)occurring at position 21.

In certain embodiments, the sense strand of the DsiRNA agent is modifiedfor Dicer processing by suitable modifiers located at the 3′ end of thesense strand, i.e., the DsiRNA agent is designed to direct orientationof Dicer binding and processing via sense strand modification. Suitablemodifiers include nucleotides such as deoxyribonucleotides,dideoxyribonucleotides, acyclonucleotides and the like and stericallyhindered molecules, such as fluorescent molecules and the like.Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the2′-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotidemodifiers could include 3′-deoxyadenosine (cordycepin),3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI),2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). In one embodiment,deoxyribonucleotides are used as the modifiers. When nucleotidemodifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotidemodifiers are substituted for the ribonucleotides on the 3′ end of thesense strand. When sterically hindered molecules are utilized, they areattached to the ribonucleotide at the 3′ end of the antisense strand.Thus, the length of the strand does not change with the incorporation ofthe modifiers. In another embodiment, the invention contemplatessubstituting two DNA bases in the DsiRNA agent to direct the orientationof Dicer processing of the antisense strand. In a further embodiment ofthe present invention, two terminal DNA bases are substituted for tworibonucleotides on the 3′-end of the sense strand forming a blunt end ofthe duplex on the 3′ end of the sense strand and the 5′ end of theantisense strand, and a two-nucleotide RNA overhang is located on the3′-end of the antisense strand. This is an asymmetric composition withDNA on the blunt end and RNA bases on the overhanging end. In certainembodiments of the instant invention, the modified nucleotides (e.g.,deoxyribonucleotides) of the penultimate and ultimate positions of the3′ terminus of the sense strand base pair with corresponding modifiednucleotides (e.g., deoxyribonucleotides) of the antisense strand(optionally, the penultimate and ultimate residues of the 5′ end of theantisense strand in those DsiRNA agents of the instant inventionpossessing a blunt end at the 3′ terminus of the sense strand/5′terminus of the antisense strand).

The sense and antisense strands of a DsiRNA agent of the instantinvention anneal under biological conditions, such as the conditionsfound in the cytoplasm of a cell. In addition, a region of one of thesequences, particularly of the antisense strand, of the DsiRNA agent hasa sequence length of at least 19 nucleotides, wherein these nucleotidesare in the 21-nucleotide region adjacent to the 3′ end of the antisensestrand and are sufficiently complementary to a nucleotide sequence ofthe RNA produced from the target gene to anneal with and/or decreaselevels of such a target RNA.

The DsiRNA agent of the instant invention may possess one or moredeoxyribonucleotide base pairs located at any positions of sense andantisense strands that are located 3′ of the projected Dicer cleavagesite of the sense strand and 5′ of the projected Dicer cleavage site ofthe antisense strand. In certain embodiments, one, two, three or allfour of positions 24-27 of the sense strand (starting from position 1 atthe 5′ terminus of the sense strand) are deoxyribonucleotides, eachdeoxyribonucleotide of which base pairs with a correspondingdeoxyribonucleotide of the antisense strand. In certain embodiments, thedeoxyribonucleotides of the 5′ region of the antisense strand (e.g., theregion of the antisense strand located 5′ of the projected Dicercleavage site for a given DsiRNA molecule) are not complementary to thetarget RNA to which the DsiRNA agent is directed. In relatedembodiments, the entire region of the antisense strand located 5′ of theprojected Dicer cleavage site of a DsiRNA agent is not complementary tothe target RNA to which the DsiRNA agent is directed. In certainembodiments, the deoxyribonucleotides of the antisense strand or theentire region of the antisense strand that is located 5′ of theprojected Dicer cleavage site of the DsiRNA agent is not sufficientlycomplementary to the target RNA to enhance annealing of the antisensestrand of the DsiRNA to the target RNA when the antisense strand isannealed to the target RNA under conditions sufficient to allow forannealing between the antisense strand and the target RNA (e.g., a“core” antisense strand sequence lacking the DNA-extended region annealsequally well to the target RNA as the same “core” antisense strandsequence also extended with sequence of the DNA-extended region).

The DsiRNA agent may also have one or more of the following additionalproperties: (a) the antisense strand has a right or left shift from thetypical 21mer, (b) the strands may not be completely complementary,i.e., the strands may contain simple mismatch pairings and (c) basemodifications such as locked nucleic acid(s) may be included in the 5′end of the sense strand. A “typical” 21mer siRNA is designed usingconventional techniques. In one technique, a variety of sites arecommonly tested in parallel or pools containing several distinct siRNAduplexes specific to the same target with the hope that one of thereagents will be effective (Ji et al., 2003). Other techniques usedesign rules and algorithms to increase the likelihood of obtainingactive RNAi effector molecules (Schwarz et al., 2003; Khvorova et al.,2003; Ui-Tei et al., 2004; Reynolds et al., 2004; Krol et al., 2004;Yuan et al., 2004; Boese et al., 2005). High throughput selection ofsiRNA has also been developed (U.S. published patent application No.2005/0042641 A1). Potential target sites can also be analyzed bysecondary structure predictions (Heale et al., 2005). This 21mer is thenused to design a right shift to include 3-9 additional nucleotides onthe 5′ end of the 21mer. The sequence of these additional nucleotidesmay have any sequence. In one embodiment, the added ribonucleotides arebased on the sequence of the target gene. Even in this embodiment, fullcomplementarity between the target sequence and the antisense siRNA isnot required.

The first and second oligonucleotides of a DsiRNA agent of the instantinvention are not required to be completely complementary. They onlyneed to be substantially complementary to anneal under biologicalconditions and to provide a substrate for Dicer that produces a siRNAsufficiently complementary to the target sequence. Locked nucleic acids,or LNA's, are well known to a skilled artisan (Elman et al., 2005;Kurreck et al., 2002; Crinelli et al., 2002; Braasch and Corey, 2001;Bondensgaard et al., 2000; Wahlestedt et al., 2000). In one embodiment,an LNA is incorporated at the 5′ terminus of the sense strand. Inanother embodiment, an LNA is incorporated at the 5′ terminus of thesense strand in duplexes designed to include a 3′ overhang on theantisense strand.

In certain embodiments, the DsiRNA agent of the instant invention has anasymmetric structure, with the sense strand having a 27-base pairlength, and the antisense strand having a 29-base pair length with a 2base 3′-overhang. Such agents optionally may possess between one andfour deoxyribonucleotides of the 3′ terminal region (specifically, theregion 3′ of the projected Dicer cleavage site) of the sense strand, atleast one of which base pairs with a cognate deoxyribonucleotide of the5′ terminal region (specifically, the region 5′ of the projected Dicercleavage site) of the antisense strand. In other embodiments, the sensestrand has a 28-base pair length, and the antisense strand has a 30-basepair length with a 2 base 3′-overhang. Such agents optionally maypossess between one and five deoxyribonucleotides of the 3′ terminalregion (specifically, the region 3′ of the projected Dicer cleavagesite) of the sense strand, at least one of which base pairs with acognate deoxyribonucleotide of the 5′ terminal region (specifically, theregion 5′ of the projected Dicer cleavage site) of the antisense strand.In additional embodiments, the sense strand has a 29-base pair length,and the antisense strand has a 31-base pair length with a 2 base3′-overhang. Such agents optionally possess between one and sixdeoxyribonucleotides of the 3′ terminal region (specifically, the region3′ of the projected Dicer cleavage site) of the sense strand, at leastone of which base pairs with a cognate deoxyribonucleotide of the 5′terminal region (specifically, the region 5′ of the projected Dicercleavage site) of the antisense strand. In further embodiments, thesense strand has a 30-base pair length, and the antisense strand has a32-base pair length with a 2 base 3′-overhang. Such agents optionallypossess between one and seven deoxyribonucleotides of the 3′ terminalregion (specifically, the region 3′ of the projected Dicer cleavagesite) of the sense strand, at least one of which base pairs with acognate deoxyribonucleotide of the 5′ terminal region (specifically, theregion 5′ of the projected Dicer cleavage site) of the antisense strand.In other embodiments, the sense strand has a 31-base pair length, andthe antisense strand has a 33-base pair length with a 2 base3′-overhang. Such agents optionally possess between one and eightdeoxyribonucleotides of the 3′ terminal region (specifically, the region3′ of the projected Dicer cleavage site) of the sense strand, at leastone of which base pairs with a cognate deoxyribonucleotide of the 5′terminal region (specifically, the region 5′ of the projected Dicercleavage site) of the antisense strand. In additional embodiments, thesense strand has a 32-base pair length, and the antisense strand has a34-base pair length with a 2 base 3′-overhang. Such agents optionallypossess between one and nine deoxyribonucleotides of the 3′ terminalregion (specifically, the region 3′ of the projected Dicer cleavagesite) of the sense strand, at least one of which base pairs with acognate deoxyribonucleotide of the 5′ terminal region (specifically, theregion 5′ of the projected Dicer cleavage site) of the antisense strand.In certain further embodiments, the sense strand has a 33-base pairlength, and the antisense strand has a 35-base pair length with a 2 base3′-overhang. Such agents optionally possess between one and tendeoxyribonucleotides of the 3′ terminal region (specifically, the region3′ of the projected Dicer cleavage site) of the sense strand, at leastone of which base pairs with a cognate deoxyribonucleotide of the 5′terminal region (specifically, the region 5′ of the projected Dicercleavage site) of the antisense strand. In still other embodiments, anyof these DsiRNA agents have an asymmetric structure that furthercontains 2 deoxyribonucleotides at the 3′ end of the sense strand inplace of two of the ribonucleotides; optionally, these 2deoxyribonucleotides base pair with cognate deoxyribonucleotides of theantisense strand.

Certain DsiRNA agent compositions containing two separateoligonucleotides can be linked by a third structure. The third structurewill not block Dicer activity on the DsiRNA agent and will not interferewith the directed destruction of the RNA transcribed from the targetgene. In one embodiment, the third structure may be a chemical linkinggroup. Many suitable chemical linking groups are known in the art andcan be used. Alternatively, the third structure may be anoligonucleotide that links the two oligonucleotides of the DsiRNA agentin a manner such that a hairpin structure is produced upon annealing ofthe two oligonucleotides making up the dsNA composition. The hairpinstructure will not block Dicer activity on the DsiRNA agent and will notinterfere with the directed destruction of the target RNA.

In certain embodiments, the DsiRNA agent of the invention has severalproperties which enhance its processing by Dicer. According to suchembodiments, the DsiRNA agent has a length sufficient such that it isprocessed by Dicer to produce an siRNA and at least one of the followingproperties: (i) the DsiRNA agent is asymmetric, e.g., has a 3′ overhangon the sense strand and (ii) the DsiRNA agent has a modified 3′ end onthe antisense strand to direct orientation of Dicer binding andprocessing of the dsRNA region to an active siRNA. According to theseembodiments, the longest strand in the DsiRNA agent comprises 25-43nucleotides. In one embodiment, the sense strand comprises 25-39nucleotides and the antisense strand comprises 26-43 nucleotides. Theresulting dsNA can have an overhang on the 3′ end of the sense strand.The overhang is 1-4 nucleotides, such as 2 nucleotides. The antisense orsense strand may also have a 5′ phosphate.

In certain embodiments, the sense strand of a DsiRNA agent is modifiedfor Dicer processing by suitable modifiers located at the 3′ end of thesense strand, i.e., the DsiRNA agent is designed to direct orientationof Dicer binding and processing. Suitable modifiers include nucleotidessuch as deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotidesand the like and sterically hindered molecules, such as fluorescentmolecules and the like. Acyclonucleotides substitute a2-hydroxyethoxymethyl group for the 2′-deoxyribofuranosyl sugar normallypresent in dNMPs. Other nucleotide modifiers could include3′-deoxyadenosine (cordycepin), 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxyinosine (ddI), 2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). In one embodiment,deoxyribonucleotides are used as the modifiers. When nucleotidemodifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotidemodifiers are substituted for the ribonucleotides on the 3′ end of thesense strand. When sterically hindered molecules are utilized, they areattached to the ribonucleotide at the 3′ end of the antisense strand.Thus, the length of the strand does not change with the incorporation ofthe modifiers. In another embodiment, the invention contemplatessubstituting two DNA bases in the dsNA to direct the orientation ofDicer processing. In a further embodiment, two terminal DNA bases arelocated on the 3′ end of the sense strand in place of tworibonucleotides forming a blunt end of the duplex on the 5′ end of theantisense strand and the 3′ end of the sense strand, and atwo-nucleotide RNA overhang is located on the 3′-end of the antisensestrand. This is an asymmetric composition with DNA on the blunt end andRNA bases on the overhanging end.

In certain other embodiments, the antisense strand of a DsiRNA agent ismodified for Dicer processing by suitable modifiers located at the 3′end of the antisense strand, i.e., the DsiRNA agent is designed todirect orientation of Dicer binding and processing. Suitable modifiersinclude nucleotides such as deoxyribonucleotides,dideoxyribonucleotides, acyclonucleotides and the like and stericallyhindered molecules, such as fluorescent molecules and the like.Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the2′-deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotidemodifiers could include 3′-deoxyadenosine (cordycepin),3′-azido-3′-deoxythymidine (AZT), 2′,3′-dideoxyinosine (ddI),2′,3′-dideoxy-3′-thiacytidine (3TC),2′,3′-didehydro-2′,3′-dideoxythymidine (d4T) and the monophosphatenucleotides of 3′-azido-3′-deoxythymidine (AZT),2′,3′-dideoxy-3′-thiacytidine (3TC) and2′,3′-didehydro-2′,3′-dideoxythymidine (d4T). In one embodiment,deoxyribonucleotides are used as the modifiers. When nucleotidemodifiers are utilized, 1-3 nucleotide modifiers, or 2 nucleotidemodifiers are substituted for the ribonucleotides on the 3′ end of theantisense strand. When sterically hindered molecules are utilized, theyare attached to the ribonucleotide at the 3′ end of the antisensestrand. Thus, the length of the strand does not change with theincorporation of the modifiers. In another embodiment, the inventioncontemplates substituting two DNA bases in the dsNA to direct theorientation of Dicer processing. In a further invention, two terminalDNA bases are located on the 3′ end of the antisense strand in place oftwo ribonucleotides forming a blunt end of the duplex on the 5′ end ofthe sense strand and the 3′ end of the antisense strand, and atwo-nucleotide RNA overhang is located on the 3′-end of the sensestrand. This is also an asymmetric composition with DNA on the blunt endand RNA bases on the overhanging end.

The sense and antisense strands anneal under biological conditions, suchas the conditions found in the cytoplasm of a cell. In addition, aregion of one of the sequences, particularly of the antisense strand, ofthe dsNA has a sequence length of at least 19 nucleotides, wherein thesenucleotides are adjacent to the 3′ end of antisense strand and aresufficiently complementary to a nucleotide sequence of the target RNA todirect RNA interference.

Additionally, the DsiRNA agent structure can be optimized to ensure thatthe oligonucleotide segment generated from Dicer's cleavage will be theportion of the oligonucleotide that is most effective in inhibiting geneexpression. For example, in one embodiment of the invention, a 27-35-bpoligonucleotide of the DsiRNA agent structure is synthesized wherein theanticipated 21 to 22-bp segment that will inhibit gene expression islocated on the 3′-end of the antisense strand. The remaining baseslocated on the 5′-end of the antisense strand will be cleaved by Dicerand will be discarded. This cleaved portion can be homologous (i.e.,based on the sequence of the target sequence) or non-homologous andadded to extend the nucleic acid strand. As surprisingly identified inthe instant invention, such extension can be performed with base pairedDNA residues (double stranded DNA:DNA extensions), resulting in extendedDsiRNA agents having improved efficacy or duration of effect thancorresponding double stranded RNA:RNA-extended DsiRNA agents.

US 2007/0265220 discloses that 27mer DsiRNAs show improved stability inserum over comparable 21mer siRNA compositions, even absent chemicalmodification. Modifications of DsiRNA agents, such as inclusion of2′-O-methyl RNA in the antisense strand, in patterns such as detailed inUS 2007/0265220 and in the instant Examples, when coupled with additionof a 5′ Phosphate, can improve stability of DsiRNA agents. Addition of5′-phosphate to all strands in synthetic RNA duplexes may be aninexpensive and physiological method to confer some limited degree ofnuclease stability.

The chemical modification patterns of the DsiRNA agents of the instantinvention are designed to enhance the efficacy of such agents.Accordingly, such modifications are designed to avoid reducing potencyof DsiRNA agents; to avoid interfering with Dicer processing of DsiRNAagents; to improve stability in biological fluids (reduce nucleasesensitivity) of DsiRNA agents; or to block or evade detection by theinnate immune system. Such modifications are also designed to avoidbeing toxic and to avoid increasing the cost or impact the ease ofmanufacturing the instant DsiRNA agents of the invention.

RNA Processing

siRNA

The process of siRNA-mediated RNAi is triggered by the presence of long,dsRNA molecules in a cell. During the initiation step of RNAi, thesedsRNA molecules are cleaved into 21-23 nucleotide (nt) small-interferingRNA duplexes (siRNAs) by Dicer, a conserved family of enzymes containingtwo RNase III-like domains (Bernstein et al. 2001; Elbashir et al.2001). The siRNAs are characterized by a 19-21 base pair duplex regionand 2 nucleotide 3′ overhangs on each strand. During the effector stepof RNAi, the siRNAs become incorporated into a multimeric proteincomplex called RNA-induced silencing complex (RISC), where they serve asguides to select fully complementary mRNA substrates for degradation.Degradation is initiated by endonucleolytic cleavage of the mRNA withinthe region complementary to the siRNA. More precisely, the mRNA iscleaved at a position 10 nucleotides from the 5′ end of the guidingsiRNA (Elbashir et al. 2001 Genes & Dev. 15: 188-200; Nykanen et al.2001 Cell 107: 309-321; Martinez et al. 2002 Cell 110: 563-574). Anendonuclease responsible for this cleavage was identified as Argonaute2(Ago2; Liu et al. Science, 305: 1437-41).

miRNA

The majority of human miRNAs (70%)—and presumably the majority of miRNAsof other mammals—are transcribed from introns and/or exons, andapproximately 30% are located in intergenic regions (Rodriguez et al.,Genome Res. 2004, 14(10A), 1902-1910). In human and animal, miRNAs areusually transcribed by RNA polymerase II (Farh et al. Science 2005,310(5755), 1817-1821), and in some cases by pol III (Borchert et al.Nat. Struct. Mol. Biol. 2006, 13(12), 1097-1101). Certain viral encodedmiRNAs are transcribed by RNA polymerase III (Pfeffer et al. Nat.Methods 2005, 2(4), 269-276; Andersson et al. J. Virol. 2005, 79(15),9556-9565), and some are located in the open reading frame of viral gene(Pfeffer et al. Nat. Methods 2005, 2(4), 269-276; Samols et al. J.Virol. 2005, 79(14), 9301-9305). miRNA transcription results in theproduction of large monocistronic, bicistronic or polycistronic primarytranscripts (pri-miRNAs). A single pri-miRNA may range fromapproximately 200 nucleotides (nt) to several kilobases (kb) in lengthand have both a 5′ 7-methylguanosine (m7) caps and a 3′ poly (A) tail.Characteristically, the mature miRNA sequences are localized to regionsof imperfect stem-loop sequences within the pri-miRNAs (Cullen, Mol.Cell 2004, 16(6), 861-865).

The first step of miRNA maturation in the nucleus is the recognition andcleavage of the pri-miRNAs by the RNase III Drosha-DGCR8 nuclearmicroprocessor complex, which releases a ˜70 nt hairpin-containingprecursor molecule called pre-miRNAs, with a monophosphate at the 5′terminus and a 2-nt overhang with a hydroxyl group at the 3′ terminus(Cai et al. RNA 2004, 10(12), 1957-1966; Lee et al. Nature 2003,425(6956), 415-419; Kim Nat. Rev. Mol. Cell. Biol. 2005, 6(5), 376-385).The next step is the nuclear transport of the pre-miRNAs out of thenucleus into the cytoplasm by Exportin-5, a carrier protein (Yi et al.Genes. Dev. 2003, 17(24), 3011-3016, Bohnsack et al. RNA 2004, 10(2),185-191). Exportin-5 and the GTP-bound form of its cofactor Ran togetherrecognize and bind the 2 nucleotide 3′ overhang and the adjacent stemthat are characteristics of pre-miRNA (Basyuk et al. Nucl. Acids Res.2003, 31(22), 6593-6597, Zamore Mol. Cell. 2001, 8(6), 1158-1160). Inthe cytoplasm, GTP hydrolysis results in release of the pre-miRNA, whichis then processed by a cellular endonuclease III enzyme Dicer (Bohnsacket al.). Dicer was first recognized for its role in generating siRNAsthat mediate RNA interference (RNAi). Dicer acts in concert with itscofactors TRBP (Transactivating region binding protein; Chendrimata etal. Nature 2005, 436(7051), 740-744) and PACT (interferon-inducibledouble strand-RNA-dependent protein kinase activator; Lee et al. EMBO J.2006, 25(3), 522-532). These enzymes bind at the 3′ 2 nucleotideoverhang at the base of the pre-miRNA hairpin and remove the terminalloop, yielding an approximately 21-nt miRNA duplex intermediate withboth termini having 5′ monophosphates, 3′ 2 nucleotide overhangs and 3′hydroxyl groups. The miRNA guide strand, the 5′ terminus of which isenergetically less stable, is then selected for incorporation into theRISC (RNA-induced silencing complex), while the ‘passenger’ strand isreleased and degraded (Maniataki et al. Genes. Dev. 2005, 19(24),2979-2990; Hammond et al. Nature 2000, 404(6775), 293-296). Thecomposition of RISC remains incompletely defined, but a key component isa member of the Argonaute (Ago) protein family (Maniataki et al.;Meister et al. Mol. Cell. 2004, 15(2), 185-197).

The mature miRNA then directs RISC to complementary mRNA species. If thetarget mRNA has perfect complementarity to the miRNA-armed RISC, themRNA will be cleaved and degraded (Zeng et al. Proc. Natl. Acad. Sci.USA 2003, 100(17), 9779-9784; Hutvagner et al. Science 2002, 297(55 89),2056-2060). But as the most common situation in mammalian cells, themiRNAs targets mRNAs with imperfect complementarity and suppress theirtranslation, resulting in reduced expression of the correspondingproteins (Yekta et al. Science 2004, 304(5670), 594-596; Olsen et al.Dev. Biol. 1999, 216(2), 671-680). The 5′ region of the miRNA,especially the match between miRNA and target sequence at nucleotides2-7 or 8 of miRNA (starting from position 1 at the 5′ terminus), whichis called the seed region, is essentially important for miRNA targeting,and this seed match has also become a key principle widely used incomputer prediction of the miRNA targeting (Lewis et al. Cell 2005,120(1), 15-20; Brennecke et al. PLoS Biol. 2005, 3(3), e85). miRNAregulation of the miRNA-mRNA duplexes is mediated mainly throughmultiple complementary sites in the 3′ UTRs, but there are manyexceptions. miRNAs may also bind the 5′ UTR and/or the coding region ofmRNAs, resulting in a similar outcome (Lytle et al. Proc. Natl. Acad.Sci. USA 2007, 104(23), 9667-9672).

RNase H

RNase H is a ribonuclease that cleaves the 3′-O—P bond of RNA in aDNA/RNA duplex to produce 3′-hydroxyl and 5′-phosphate terminatedproducts. RNase H is a non-specific endonuclease and catalyzes cleavageof RNA via a hydrolytic mechanism, aided by an enzyme-bound divalentmetal ion. Members of the RNase H family are found in nearly allorganisms, from archaea and prokaryotes to eukaryotes. During DNAreplication, RNase H is believed to cut the RNA primers responsible forpriming generation of Okazaki fragments; however, the RNase H enzyme maybe more generally employed to cleave any DNA:RNA hybrid sequence ofsufficient length (e.g., typically DNA:RNA hybrid sequences of 4 or morebase pairs in length in mammals).

MicroRNA and MicroRNA-Like Therapeutics

MicroRNAs (miRNAs) have been described to act by binding to the 3′ UTRof a template transcript, thereby inhibiting expression of a proteinencoded by the template transcript by a mechanism related to butdistinct from classic RNA interference. Specifically, miRNAs arebelieved to act by reducing translation of the target transcript, ratherthan by decreasing its stability. Naturally-occurring miRNAs aretypically approximately 22 nt in length. It is believed that they arederived from larger precursors known as small temporal RNAs (stRNAs)approximately 70 nt long.

Interference agents such as siRNAs, and more specifically such asmiRNAs, that bind within the 3′ UTR (or elsewhere in a targettranscript, e.g., in repeated elements of, e.g., Notch and/ortranscripts of the Notch family) and inhibit translation may tolerate alarger number of mismatches in the siRNA/template (miRNA/template)duplex, and particularly may tolerate mismatches within the centralregion of the duplex. In fact, there is evidence that some mismatchesmay be desirable or required, as naturally occurring stRNAs frequentlyexhibit such mismatches, as do miRNAs that have been shown to inhibittranslation in vitro (Zeng et al., Molecular Cell, 9: 1-20). Forexample, when hybridized with the target transcript, such miRNAsfrequently include two stretches of perfect complementarity separated bya region of mismatch. Such a hybridized complex commonly includes tworegions of perfect complementarily (duplex portions) comprisingnucleotide pairs, and at least a single mismatched base pair, which maybe, e.g., G:A, G:U, G:G, A:A, A:C, U:U, U:C, C:C, G:-, A:-, U:-, C:-,etc. Such mismatched nucleotides, especially if present in tandem (e.g.,a two, three or four nucleotide area of mismatch) can form a bulge thatseparates duplex portions which are located on either flank of such abulge. A variety of structures are possible. For example, the miRNA mayinclude multiple areas of nonidentity (mismatch). The areas ofnonidentity (mismatch) need not be symmetrical in the sense that boththe target and the miRNA include nonpaired nucleotides. For example,structures have been described in which only one strand includesnonpaired nucleotides (Zeng et al.). Typically the stretches of perfectcomplementarily within a miRNA agent are at least 5 nucleotides inlength, e.g., 6, 7, or more nucleotides in length, while the regions ofmismatch may be, for example, 1, 2, 3, or 4 nucleotides in length.

In general, any particular siRNA could function to inhibit geneexpression both via (i) the “classical” siRNA pathway, in whichstability of a target transcript is reduced and in which perfectcomplementarily between the siRNA and the target is frequentlypreferred, and also by (ii) the “alternative” pathway (generallycharacterized as the miRNA pathway in animals), in which translation ofa target transcript is inhibited. Generally, the transcripts targeted bya particular siRNA via mechanism (i) would be distinct from thetranscript targeted via mechanism (ii), although it is possible that asingle transcript could contain regions that could serve as targets forboth the classical and alternative pathways. (Note that the terms“classical” and “alternative” are used merely for convenience andgenerally are believed to reflect historical timing of discovery of suchmechanisms in animal cells, but do not reflect the importance,effectiveness, or other features of either mechanism.) One common goalof siRNA design has been to target a single transcript with greatspecificity, via mechanism (i), while minimizing off-target effects,including those effects potentially elicited via mechansim (ii).However, it is among the goals of the instant invention to provide RNAinterference agents that possess mismatch residues by design, either forpurpose of mimicking the activities of naturally-occurring miRNAs, or tocreate agents directed against target RNAs for which no correspondingmiRNA is presently known, with the inhibitory and/or therapeuticefficacies/potencies of such mismatch-containing DsiRNA agents (e.g.,DsiRNAmm agents) tolerant of, and indeed possibly enhanced by, suchmismatches.

The tolerance of miRNA agents for mismatched nucleotides (and, indeedthe existence and natural use of mechanism (ii) above in the cell)suggests the use of miRNAs in manners that are advantageous to and/orexpand upon the “classical” use of perfectly complementary siRNAs thatact via mechanism (i). Because miRNAs are naturally occurring molecules,there are likely to be distinct advantages in applying miRNAs astherapeutic agents. miRNAs benefit from hundreds of millions of years ofevolutionary “fine tuning” of their function. Thus, sequence-specific“off target.” effects should not be an issue with naturally occurringmiRNAs, nor, by extension, with certain synthetic DsiRNAs of theinvention (e.g., DsiRNAmm agents) designed to mimic naturally occurringmRNAs. In addition, miRNAs have evolved to modulate the expression ofgroups of genes, driving both up and down regulation (in certaininstances, performing both functions concurrently within a cell with asingle miRNA acting promiscuously upon multiple target RNAs), with theresult that complex cell functions can be precisely modulated, Suchreplacement of naturally occurring miRNAs can involve introducingsynthetic miRNAs or miRNA mimetics (e.g., certain DsiRNAmms) intodiseased tissues in an effort to restore normal proliferation,apoptosis, cell cycle, and other cellular functions that have beenaffected by down-regulation of one or more miRNAs. In certain instances,reactivation of these miRNA-regulated pathways has produced asignificant therapeutic response (e.g., In one study on cardiachypertrophy, overexpression of miR-133 by adenovirus-mediated deliveryof a miRNA expression cassette protected animals from agonist-inducedcardiac hypertrophy, whereas reciprocally reduction of miR-133 inwild-type mice by antagomirs caused an increase in hypertrophic markers(Care et al. Nat. Med. 13: 613-618)).

To date, more than 600 miRNAs have been identified as encoded within thehuman genome, with such miRNAs expressed and processed by a combinationof proteins in the nucleus and cytoplasm. miRNAs are highly conservedamong vertebrates and comprise approximately 2% of all mammalian genes.Since each miRNA appears to regulate the expression of multiple, e.g.,two, three, four, five, six, seven, eight, nine or even tens to hundredsof different genes, miRNAs can function as “master-switches”,efficiently regulating and coordinating multiple cellular pathways andprocesses. By coordinating the expression of multiple genes, miRNAs playkey roles in embryonic development, immunity, inflammation, as well ascellular growth and proliferation.

Expression and functional studies suggest that the altered expression ofspecific miRNAs is critical to a variety of human diseases. Mountingevidence indicates that the introduction of specific miRNAs into diseasecells and tissues can induce favorable therapeutic responses (Pappas etal., Expert Opin Ther Targets. 12: 115-27). The promise of miRNA therapyis perhaps greatest in cancer due to the apparent role of certain miRNAsas tumor suppressors. The rationale for miRNA-based therapeutics for,e.g., cancer is supported, at least in part, by the followingobservations:

-   -   (1) miRNAs are frequently mis-regulated and expressed at altered        levels in diseased tissues when compared to normal tissues. A        number of studies have shown altered levels of miRNAs in        cancerous tissues relative to their corresponding normal        tissues. Often, altered expression is the consequence of genetic        mutations that lead to increased or reduced expression of        particular miRNAs. Diseases that possess unique miRNA expression        signatures can be exploited as diagnostic and prognostic        markers, and can be targeted with the DsiRNA (e.g., DsiRNAmm)        agents of the invention.    -   (2) Mis-regulated miRNAs contribute to cancer development by        functioning as oncogenes or tumor suppressors. Oncogenes are        defined as genes whose over-expression or inappropriate        activation leads to oncogenesis. Tumor suppressors are genes        that are required to keep cells from being cancerous; the        down-regulation or inactivation of tumor suppressors is a common        inducer of cancer. Both types of genes represent preferred drug        targets, as such targeting can specifically act upon the        molecular basis for a particular cancer. Examples of oncogenic        miRNAs are miR-155 and miR-17-92; let-7 is an example of a tumor        suppressive miRNA.    -   (3) Administration of miRNA induces a therapeutic response by        blocking or reducing tumor growth in pre-clinical animal        studies. The scientific literature provides proof-of-concept        studies demonstrating that restoring miRNA function can prevent        or reduce the growth of cancer cells in vitro and also in animal        models. A well-characterized example is the anti-tumor activity        of let-7 in models for breast and lung cancer. DsiRNAs (e.g.,        DsiRNAmms) of the invention which are designed to mimic let-7        can be used to target such cancers, and it is also possible to        use the DsiRNA design parameters described herein to generate        new DsiRNA (e.g., DsiRNAmm) agents directed against target RNAs        for which no counterpart naturally occurring miRNA is known        (e.g., repeats within Notch or other transcripts), to screen for        therapeutic lead compounds, e.g., agents that are capable of        reducing tumor burden in pre-clinical animal models.    -   (4) A given miRNA controls multiple cellular pathways and        therefore may have superior therapeutic activity. Based on their        biology, miRNAs can function as “master switches” of the genome,        regulating multiple gene products and coordinating multiple        pathways. Genes regulated by miRNAs include genes that encode        conventional oncogenes and tumor suppressors, many of which are        individually pursued as drug targets by the pharmaceutical        industry. Thus, miRNA therapeutics could possess activity        superior to siRNAs and other forms of lead compounds by        targeting multiple disease and/or cancer-associated genes. Given        the observation that mis-regulation of miRNAs is frequently an        early event in the process of tumorigenesis, miRNA therapeutics,        which replace missing miRNAs, may be the most appropriate        therapy.    -   (5) miRNAs are natural molecules and are therefore less prone to        induce non-specific side-effects. Millions of years of evolution        helped to develop the regulatory network of miRNAs, fine-tuning        the interaction of miRNA with target messenger RNAs. Therefore,        miRNAs and miRNA derivatives (e.g., DsiRNAs designed to mimic        naturally occurring miRNAs) will have few if any        sequence-specific “off-target” effects when applied in the        proper context.

The physical characteristics of siRNAs and miRNAs are similar.Accordingly, technologies that are effective in delivering siRNAs (e.g.,DsiRNAs of the invention) are likewise effective in delivering syntheticmiRNAs (e.g., certain DsiRNAmms of the invention).

Conjugation and Delivery of DsiRNA Agents

In certain embodiments, the present invention relates to a method fortreating a subject having or at risk of developing a disease ordisorder. In such embodiments, the DsiRNA can act as a novel therapeuticagent for controlling the disease or disorder. The method comprisesadministering a pharmaceutical composition of the invention to thepatient (e.g., human), such that the expression, level and/or activity atarget RNA is reduced. The expression, level and/or activity of apolypeptide encoded by the target RNA might also be reduced by a DsiRNAof the instant invention.

In the treatment of a disease or disorder, the DsiRNA can be broughtinto contact with the cells or tissue exhibiting or associated with adisease or disorder. For example, DsiRNA substantially identical to allor part of a target RNA sequence, may be brought into contact with orintroduced into a diseased, disease-associated or infected cell, eitherin vivo or in vitro. Similarly, DsiRNA substantially identical to all orpart of a target RNA sequence may administered directly to a subjecthaving or at risk of developing a disease or disorder.

Therapeutic use of the DsiRNA agents of the instant invention caninvolve use of formulations of DsiRNA agents comprising multipledifferent DsiRNA agent sequences. For example, two or more, three ormore, four or more, five or more, etc. of the presently described agentscan be combined to produce a formulation that, e.g., targets multipledifferent regions of one or more target RNA(s). A DsiRNA agent of theinstant invention may also be constructed such that either strand of theDsiRNA agent independently targets two or more regions of a target RNA.Use of multifunctional DsiRNA molecules that target more then one regionof a target nucleic acid molecule is expected to provide potentinhibition of RNA levels and expression. For example, a singlemultifunctional DsiRNA construct of the invention can target bothconserved and variable regions of a target nucleic acid molecule,thereby allowing down regulation or inhibition of, e.g., differentstrain variants of a virus, or splice variants encoded by a singletarget gene.

A DsiRNA agent of the invention can be conjugated (e.g., at its 5′ or 3′terminus of its sense or antisense strand) or unconjugated to anothermoiety (e.g. a non-nucleic acid moiety such as a peptide), an organiccompound (e.g., a dye, cholesterol, or the like). Modifying DsiRNAagents in this way may improve cellular uptake or enhance cellulartargeting activities of the resulting DsiRNA agent derivative ascompared to the corresponding unconjugated DsiRNA agent, are useful fortracing the DsiRNA agent derivative in the cell, or improve thestability of the DsiRNA agent derivative compared to the correspondingunconjugated DsiRNA agent.

RNAi In Vitro Assay to Assess DsiRNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system canoptionally be used to evaluate DsiRNA constructs. For example, such anassay comprises a system described by Tuschl et al., 1999, Genes andDevelopment, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33,adapted for use with DsiRNA agents directed against target RNA, andcommercially available kits, including Turbo Dicer (Genlantis). ADrosophila extract derived from syncytial blastoderm is used toreconstitute RNAi activity in vitro. Target RNA is generated via invitro transcription from an appropriate plasmid using T7 RNA polymeraseor via chemical synthesis. Sense and antisense DsiRNA strands (forexample 20 uM each) are annealed by incubation in buffer (such as 100 mMpotassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for1 minute at 90° C. followed by 1 hour at 37° C., then diluted in lysisbuffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4,2 mM magnesium acetate). Annealing can be monitored by gelelectrophoresis on an agarose gel in TBE buffer and stained withethidium bromide. The Drosophila lysate is prepared using zero totwo-hour-old embryos from Oregon R flies collected on yeasted molassesagar that are dechorionated and lysed. The lysate is centrifuged and thesupernatant isolated. The assay comprises a reaction mixture containing50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10%[vol/vol] lysis buffer containing DsiRNA (10 nM final concentration).The reaction mixture also contains 10 mM creatine phosphate, 10 ug/mlcreatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP,5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acid. Thefinal concentration of potassium acetate is adjusted to 100 mM. Thereactions are pre-assembled on ice and preincubated at 25° C. for 10minutes before adding RNA, then incubated at 25° C. for an additional 60minutes. Reactions are quenched with 4 volumes of 1.25×Passive LysisBuffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis orother methods known in the art and are compared to control reactions inwhich DsiRNA is omitted from the reaction.

Alternately, internally-labeled target RNA for the assay is prepared byin vitro transcription in the presence of [alpha-32P] CTP, passed over aG50 Sephadex column by spin chromatography and used as target RNAwithout further purification. Optionally, target RNA is 5′-32P-endlabeled using T4 polynucleotide kinase enzyme. Assays are performed asdescribed above and target RNA and the specific RNA cleavage productsgenerated by RNAi are visualized on an autoradiograph of a gel. Thepercentage of cleavage is determined by PHOSPHOR IMAGER®(autoradiography) quantitation of bands representing intact control RNAor RNA from control reactions without DsiRNA and the cleavage productsgenerated by the assay.

Methods of Introducing Nucleic Acids, Vectors, and Host Cells

DsiRNA agents of the invention may be directly introduced into a cell(i.e., intracellularly); or introduced extracellularly into a cavity,interstitial space, into the circulation of an organism, introducedorally, or may be introduced by bathing a cell or organism in a solutioncontaining the nucleic acid. Vascular or extravascular circulation, theblood or lymph system, and the cerebrospinal fluid are sites where thenucleic acid may be introduced.

The DsiRNA agents of the invention can be introduced using nucleic aciddelivery methods known in art including injection of a solutioncontaining the nucleic acid, bombardment by particles covered by thenucleic acid, soaking the cell or organism in a solution of the nucleicacid, or electroporation of cell membranes in the presence of thenucleic acid. Other methods known in the art for introducing nucleicacids to cells may be used, such as lipid-mediated carrier transport,chemical-mediated transport, and cationic liposome transfection such ascalcium phosphate, and the like. The nucleic acid may be introducedalong with other components that perform one or more of the followingactivities: enhance nucleic acid uptake by the cell or other-wiseincrease inhibition of the target RNA.

A cell having a target RNA may be from the germ line or somatic,totipotent or pluripotent, dividing or non-dividing, parenchyma orepithelium, immortalized or transformed, or the like. The cell may be astem cell or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

Depending on the particular target RNA sequence and the dose of DsiRNAagent material delivered, this process may provide partial or completeloss of function for the target RNA. A reduction or loss of RNA levelsor expression (either RNA expression or encoded polypeptide expression)in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more of targetedcells is exemplary. Inhibition of target RNA levels or expression refersto the absence (or observable decrease) in the level of RNA orRNA-encoded protein. Specificity refers to the ability to inhibit thetarget RNA without manifest effects on other genes of the cell. Theconsequences of inhibition can be confirmed by examination of theoutward properties of the cell or organism (as presented below in theexamples) or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, enzyme linked immunosorbent assay (ELISA), Western blotting,radioimmunoassay (RIA), other immunoassays, and fluorescence activatedcell analysis (FACS). Inhibition of target RNA sequence(s) by the DsiRNAagents of the invention also can be measured based upon the effect ofadministration of such DsiRNA agents upon measurable phenotypes such astumor size for cancer treatment, viral load/titer for viral infectiousdiseases, etc. either in vivo or in vitro. For viral infectiousdiseases, reductions in viral load or titer can include reductions of,e.g., 50%, 60%, 70%, 80%, 90%, 95% or 99% or more, and are oftenmeasured in logarithmic terms, e.g., 10-fold, 100-fold, 1000-fold,10⁵-fold, 10⁶-fold, 10⁷-fold reduction in viral load or titer can beachieved via administration of the DsiRNA agents of the invention tocells, a tissue, or a subject.

For RNA-mediated inhibition in a cell line or whole organism, expressiona reporter or drug resistance gene whose protein product is easilyassayed can be measured. Such reporter genes include acetohydroxyacidsynthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ),beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), greenfluorescent protein (GFP), horseradish peroxidase (HRP), luciferase(Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivativesthereof. Multiple selectable markers are available that conferresistance to ampicillin, bleomycin, chloramphenicol, gentarnycin,hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin,puromycin, and tetracyclin. Depending on the assay, quantitation of theamount of gene expression allows one to determine a degree of inhibitionwhich is greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to acell not treated according to the present invention.

Lower doses of injected material and longer times after administrationof RNA silencing agent may result in inhibition in a smaller fraction ofcells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of targetedcells). Quantitation of gene expression in a cell may show similaramounts of inhibition at the level of accumulation of target RNA ortranslation of target protein. As an example, the efficiency ofinhibition may be determined by assessing the amount of gene product inthe cell; RNA may be detected with a hybridization probe having anucleotide sequence outside the region used for the inhibitory DsiRNA,or translated polypeptide may be detected with an antibody raisedagainst the polypeptide sequence of that region.

The DsiRNA agent may be introduced in an amount which allows delivery ofat least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500or 1000 copies per cell) of material may yield more effectiveinhibition; lower doses may also be useful for specific applications.

RNA Interference Based Therapy

As is known, RNAi methods are applicable to a wide variety of genes in awide variety of organisms and the disclosed compositions and methods canbe utilized in each of these contexts. Examples of genes which can betargeted by the disclosed compositions and methods include endogenousgenes which are genes that are native to the cell or to genes that arenot normally native to the cell. Without limitation, these genes includeoncogenes, cytokine genes, idiotype (Id) protein genes, prion genes,genes that expresses molecules that induce angiogenesis, genes foradhesion molecules, cell surface receptors, proteins involved inmetastasis, proteases, apoptosis genes, cell cycle control genes, genesthat express EGF and the EGF receptor, multi-drug resistance genes, suchas the MDR1 gene.

More specifically, a target mRNA of the invention can specify the aminoacid sequence of a cellular protein (e.g., a nuclear, cytoplasmic,transmembrane, or membrane-associated protein). In another embodiment,the target mRNA of the invention can specify the amino acid sequence ofan extracellular protein (e.g., an extracellular matrix protein orsecreted protein). As used herein, the phrase “specifies the amino acidsequence” of a protein means that the mRNA sequence is translated intothe amino acid sequence according to the rules of the genetic code. Thefollowing classes of proteins are listed for illustrative purposes:developmental proteins (e.g., adhesion molecules, cyclin kinaseinhibitors, Wnt family members, Pax family members, Winged helix familymembers, Hox family members, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, neurotransmittersand their receptors); oncogene-encoded proteins (e.g., ABLI, BCLI, BCL2,BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6, FGR, FOS,FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN,NRAS, PIM I, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressorproteins (e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF I, NF2, RB I, TP53,and WTI); and enzymes (e.g., ACC synthases and oxidases, ACP desaturasesand hydroxylases, ADP-glucose pyrophorylases, ATPases, alcoholdehydrogenases, amylases, amyloglucosidases, catalases, cellulases,chalcone synthases, chitinases, cyclooxygenases, decarboxylases,dextriinases, DNA and RNA polymerases, galactosidases, glucanases,glucose oxidases, granule-bound starch synthases, GTPases, helicases,hernicellulases, integrases, inulinases, invertases, isomerases,kinases, lactases, lipases, lipoxygenases, lysozymes, nopalinesynthases, octopine synthases, pectinesterases, peroxidases,phosphatases, phospholipases, phosphorylases, phytases, plant growthregulator synthases, polygalacturonases, proteinases and peptidases,pullanases, recombinases, reverse transcriptases, RUBISCOs,topoisomerases, and xylanases).

In one aspect, the target mRNA molecule of the invention specifies theamino acid sequence of a protein associated with a pathologicalcondition. For example, the protein may be a pathogen-associated protein(e.g., a viral protein involved in immunosuppression of the host,replication of the pathogen, transmission of the pathogen, ormaintenance of the infection), or a host protein which facilitates entryof the pathogen into the host, drug metabolism by the pathogen or host,replication or integration of the pathogen's genome, establishment orspread of infection in the host, or assembly of the next generation ofpathogen. Pathogens include RNA viruses such as flaviviruses,picornaviruses, rhabdoviruses, filoviruses, retroviruses, includinglentiviruses, or DNA viruses such as adenoviruses, poxviruses, herpesviruses, cytomegaloviruses, hepadnaviruses or others. Additionalpathogens include bacteria, fungi, helminths, schistosomes andtrypanosomes. Other kinds of pathogens can include mammaliantransposable elements. Alternatively, the protein may be atumor-associated protein or an autoimmune disease-associated protein.

The target gene may be derived from or contained in any organism. Theorganism may be a plant, animal, protozoa, bacterium, virus or fungus.See e.g., U.S. Pat. No. 6,506,559, incorporated herein by reference.

Pharmaceutical Compositions

In certain embodiments, the present invention provides for apharmaceutical composition comprising the DsiRNA agent of the presentinvention. The DsiRNA agent sample can be suitably formulated andintroduced into the environment of the cell by any means that allows fora sufficient portion of the sample to enter the cell to induce genesilencing, if it is to occur. Many formulations for dsNA are known inthe art and can be used so long as the dsNA gains entry to the targetcells so that it can act. See, e.g., U.S. published patent applicationNos. 2004/0203145 A1 and 2005/0054598 A1. For example, the DsiRNA agentof the instant invention can be formulated in buffer solutions such asphosphate buffered saline solutions, liposomes, micellar structures, andcapsids. Formulations of DsiRNA agent with cationic lipids can be usedto facilitate transfection of the DsiRNA agent into cells. For example,cationic lipids, such as lipofectin (U.S. Pat. No. 5,705,188), cationicglycerol derivatives, and polycationic molecules, such as polylysine(published PCT International Application WO 97/30731), can be used.Suitable lipids include Oligofectamine, Lipofectamine (LifeTechnologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.),or FuGene 6 (Roche) all of which can be used according to themanufacturer's instructions.

Such compositions typically include the nucleic acid molecule and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” includes saline, solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. Supplementary active compounds can alsobe incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle, which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer. Such methods include those described in U.S. Pat. No.6,468,798.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

The compounds can also be administered by transfection or infectionusing methods known in the art, including but not limited to the methodsdescribed in McCaffrey et al. (2002), Nature, 418(6893), 38-9(hydrodynamic transfection); Xia et al. (2002), Nature Biotechnol.,20(10), 1006-10 (viral-mediated delivery); or Putnam (1996), Am. J.Health Syst. Pharm. 53(2), 151-160, erratum at Am. J. Health Syst.Pharm. 53(3), 325 (1996).

The compounds can also be administered by any method suitable foradministration of nucleic acid agents, such as a DNA vaccine. Thesemethods include gene guns, bio injectors, and skin patches as well asneedle-free methods such as the micro-particle DNA vaccine technologydisclosed in U.S. Pat. No. 6,194,389, and the mammalian transdermalneedle-free vaccination with powder-form vaccine as disclosed in U.S.Pat. No. 6,168,587. Additionally, intranasal delivery is possible, asdescribed in, inter alia, Hamajima et al. (1998), Clin. Immunol.Immunopathol., 88(2), 205-10. Liposomes (e.g., as described in U.S. Pat.No. 6,472,375) and microencapsulation can also be used. Biodegradabletargetable microparticle delivery systems can also be used (e.g., asdescribed in U.S. Pat. No. 6,471,996).

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Suchformulations can be prepared using standard techniques. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i. e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of a nucleic acidmolecule (i.e., an effective dosage) depends on the nucleic acidselected. For instance, if a plasmid encoding a DsiRNA agent isselected, single dose amounts in the range of approximately 1 pg to 1000mg may be administered; in some embodiments, 10, 30, 100, or 1000 pg, or10, 30, 100, or 1000 ng, or 10, 30, 100, or 1000 μg, or 10, 30, 100, or1000 mg may be administered. In some embodiments, 1-5 g of thecompositions can be administered. The compositions can be administeredfrom one or more times per day to one or more times per week; includingonce every other day. The skilled artisan will appreciate that certainfactors may influence the dosage and timing required to effectivelytreat a subject, including but not limited to the severity of thedisease or disorder, previous treatments, the general health and/or ageof the subject, and other diseases present. Moreover, treatment of asubject with a therapeutically effective amount of a protein,polypeptide, or antibody can include a single treatment or, preferably,can include a series of treatments.

It can be appreciated that the method of introducing DsiRNA agents intothe environment of the cell will depend on the type of cell and the makeup of its environment. For example, when the cells are found within aliquid, one preferable formulation is with a lipid formulation such asin lipofectamine and the DsiRNA agents can be added directly to theliquid environment of the cells. Lipid formulations can also beadministered to animals such as by intravenous, intramuscular, orintraperitoneal injection, or orally or by inhalation or other methodsas are known in the art. When the formulation is suitable foradministration into animals such as mammals and more specificallyhumans, the formulation is also pharmaceutically acceptable.Pharmaceutically acceptable formulations for administeringoligonucleotides are known and can be used. In some instances, it may bepreferable to formulate DsiRNA agents in a buffer or saline solution anddirectly inject the formulated DsiRNA agents into cells, as in studieswith oocytes. The direct injection of DsiRNA agents duplexes may also bedone. For suitable methods of introducing dsNA (e.g., DsiRNA agents),see U.S. published patent application No. 2004/0203145 A1.

Suitable amounts of a DsiRNA agent must be introduced and these amountscan be empirically determined using standard methods. Typically,effective concentrations of individual DsiRNA agent species in theenvironment of a cell will be about 50 nanomolar or less, 10 nanomolaror less, or compositions in which concentrations of about 1 nanomolar orless can be used. In another embodiment, methods utilizing aconcentration of about 200 picomolar or less, and even a concentrationof about 50 picomolar or less, about 20 picomolar or less, about 10picomolar or less, or about 5 picomolar or less can be used in manycircumstances.

The method can be carried out by addition of the DsiRNA agentcompositions to any extracellular matrix in which cells can liveprovided that the DsiRNA agent composition is formulated so that asufficient amount of the DsiRNA agent can enter the cell to exert itseffect. For example, the method is amenable for use with cells presentin a liquid such as a liquid culture or cell growth media, in tissueexplants, or in whole organisms, including animals, such as mammals andespecially humans.

The level or activity of a target RNA can be determined by any suitablemethod now known in the art or that is later developed. It can beappreciated that the method used to measure a target RNA and/or theexpression of a target RNA can depend upon the nature of the target RNA.For example, if the target RNA encodes a protein, the term “expression”can refer to a protein or the RNA/transcript derived from the targetRNA. In such instances, the expression of a target RNA can be determinedby measuring the amount of RNA corresponding to the target RNA or bymeasuring the amount of that protein. Protein can be measured in proteinassays such as by staining or immunoblotting or, if the proteincatalyzes a reaction that can be measured, by measuring reaction rates.All such methods are known in the art and can be used. Where target RNAlevels are to be measured, any art-recognized methods for detecting RNAlevels can be used (e.g., RT-PCR, Northern Blotting, etc.). In targetingviral RNAs with the DsiRNA agents of the instant invention, it is alsoanticipated that measurement of the efficacy of a DsiRNA agent inreducing levels of a target virus in a subject, tissue, in cells, eitherin vitro or in vivo, or in cell extracts can also be used to determinethe extent of reduction of target viral RNA level(s). Any of the abovemeasurements can be made on cells, cell extracts, tissues, tissueextracts or any other suitable source material.

The determination of whether the expression of a target RNA has beenreduced can be by any suitable method that can reliably detect changesin RNA levels. Typically, the determination is made by introducing intothe environment of a cell undigested DsiRNA such that at least a portionof that DsiRNA agent enters the cytoplasm, and then measuring the levelof the target RNA. The same measurement is made on identical untreatedcells and the results obtained from each measurement are compared.

The DsiRNA agent can be formulated as a pharmaceutical composition whichcomprises a pharmacologically effective amount of a DsiRNA agent andpharmaceutically acceptable carrier. A pharmacologically ortherapeutically effective amount refers to that amount of a DsiRNA agenteffective to produce the intended pharmacological, therapeutic orpreventive result. The phrases “pharmacologically effective amount” and“therapeutically effective amount” or simply “effective amount” refer tothat amount of an RNA effective to produce the intended pharmacological,therapeutic or preventive result. For example, if a given clinicaltreatment is considered effective when there is at least a 20% reductionin a measurable parameter associated with a disease or disorder, atherapeutically effective amount of a drug for the treatment of thatdisease or disorder is the amount necessary to effect at least a 20%reduction in that parameter.

Suitably formulated pharmaceutical compositions of this invention can beadministered by any means known in the art such as by parenteral routes,including intravenous, intramuscular, intraperitoneal, subcutaneous,transdermal, airway (aerosol), rectal, vaginal and topical (includingbuccal and sublingual) administration. In some embodiments, thepharmaceutical compositions are administered by intravenous orintraparenteral infusion or injection.

In general, a suitable dosage unit of dsNA will be in the range of 0.001to 0.25 milligrams per kilogram body weight of the recipient per day, orin the range of 0.01 to 20 micrograms per kilogram body weight per day,or in the range of 0.01 to 10 micrograms per kilogram body weight perday, or in the range of 0.10 to 5 micrograms per kilogram body weightper day, or in the range of 0.1 to 2.5 micrograms per kilogram bodyweight per day. Pharmaceutical composition comprising the dsNA can beadministered once daily. However, the therapeutic agent may also bedosed in dosage units containing two, three, four, five, six or moresub-doses administered at appropriate intervals throughout the day. Inthat case, the dsNA contained in each sub-dose must be correspondinglysmaller in order to achieve the total daily dosage unit. The dosage unitcan also be compounded for a single dose over several days, e.g., usinga conventional sustained release formulation which provides sustainedand consistent release of the dsNA over a several day period. Sustainedrelease formulations are well known in the art. In this embodiment, thedosage unit contains a corresponding multiple of the daily dose.Regardless of the formulation, the pharmaceutical composition mustcontain dsNA in a quantity sufficient to inhibit expression of thetarget gene in the animal or human being treated. The composition can becompounded in such a way that the sum of the multiple units of dsNAtogether contain a sufficient dose.

Data can be obtained from cell culture assays and animal studies toformulate a suitable dosage range for humans. The dosage of compositionsof the invention lies within a range of circulating concentrations thatinclude the ED₅₀ (as determined by known methods) with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the compound that includes the IC₅₀ (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsof dsNA in plasma may be measured by standard methods, for example, byhigh performance liquid chromatography.

The pharmaceutical compositions can be included in a kit, container,pack, or dispenser together with instructions for administration.

Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a diseaseor disorder caused, in whole or in part, by the expression of a targetRNA and/or the presence of such target RNA (e.g., in the context of aviral infection, the presence of a target RNA of the viral genome,capsid, host cell component, etc.).

“Treatment”, or “treating” as used herein, is defined as the applicationor administration of a therapeutic agent (e.g., a DsiRNA agent or vectoror transgene encoding same) to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has the disease or disorder, a symptom of disease ordisorder or a predisposition toward a disease or disorder, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve or affect the disease or disorder, the symptoms of the diseaseor disorder, or the predisposition toward disease.

In one aspect, the invention provides a method for preventing in asubject, a disease or disorder as described above, by administering tothe subject a therapeutic agent (e.g., a DsiRNA agent or vector ortransgene encoding same). Subjects at risk for the disease can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the detection of, e.g., viral particles in asubject, or the manifestation of symptoms characteristic of the diseaseor disorder, such that the disease or disorder is prevented or,alternatively, delayed in its progression.

Another aspect of the invention pertains to methods of treating subjectstherapeutically, i.e., alter onset of symptoms of the disease ordisorder. These methods can be performed in vitro (e.g., by culturingthe cell with the DsiRNA agent) or, alternatively, in vivo (e.g., byadministering the DsiRNA agent to a subject).

With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the target RNAmolecules of the present invention or target RNA modulators according tothat individual's drug response genotype. Pharmacogenomics allows aclinician or physician to target prophylactic or therapeutic treatmentsto patients who will most benefit from the treatment and to avoidtreatment of patients who will experience toxic drug-related sideeffects.

Therapeutic agents can be tested in an appropriate animal model. Forexample, a DsiRNA agent (or expression vector or transgene encodingsame) as described herein can be used in an animal model to determinethe efficacy, toxicity, or side effects of treatment with said agent.Alternatively, a therapeutic agent can be used in an animal model todetermine the mechanism of action of such an agent. For example, anagent can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent can be used in an animal model to determine themechanism of action of such an agent.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA, genetics, immunology, cell biology, cellculture and transgenic biology, which are within the skill of the art.See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.); Sambrook et al., 1989,Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Sambrook and Russell, 2001, Molecular Cloning, 3rdEd. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.);Ausubel et al., 1992), Current Protocols in Molecular Biology (JohnWiley & Sons, including periodic updates); Glover, 1985, DNA Cloning(IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow andLane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.); Jakoby and Pastan, 1979; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Riott, Essential Immunology, 6thEdition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al.,Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1986); Westerfield, M., The zebrafish book. Aguide for the laboratory use of zebrafish (Danio rerio), (4th Ed., Univ.of Oregon Press, Eugene, 2000).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Examples

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described below were utilized.

Example 1—Methods

Oligonucleotide synthesis, In Vitro Use

Individual RNA strands were synthesized and HPLC purified according tostandard methods (Integrated DNA Technologies, Coralville, Iowa). Alloligonucleotides were quality control released on the basis of chemicalpurity by HPLC analysis and full length strand purity by massspectrometry analysis. Duplex RNA DsiRNAs were prepared before use bymixing equal quantities of each strand, briefly heating to 100° C. inRNA buffer (IDT) and then allowing the mixtures to cool to roomtemperature.

Oligonucleotide synthesis, In Vivo Use

Individual RNA strands were synthesized and HPLC purified according tostandard methods (OligoFactory, Holliston, Mass.). All oligonucleotideswere quality control released on the basis of chemical purity by HPLCanalysis and full length strand purity by mass spectrometry analysis.Duplex RNA DsiRNAs were prepared before use by mixing equimolarquantities of each strand, briefly heating to 100° C. in RNA buffer(IDT) and then allowing the mixtures to cool to room temperature.

Cell Culture and RNA Transfection

HeLa cells were obtained from ATCC and maintained in Dulbecco's modifiedEagle medium (HyClone) supplemented with 10% fetal bovine serum(HyClone) at 37° C. under 5% CO₂. For RNA transfections of FIGS. 7, 9,12, and 13, HeLa cells were transfected with DsiRNAs as indicated at afinal concentration of 0.1 nM using Lipofectamine™ RNAiMAX (Invitrogen)and following manufacturer's instructions. Briefly, 2.5 μL of a 0.02 μMstock solution of each DsiRNA were mix with 46.5 μL of Opti-MEM I(Invitrogen) and 1 μL of Lipofectamine™ RNAiMAX. The resulting 50 μL mixwas added into individual wells of 12 well plates and incubated for 20min at RT to allow DsiRNA:Lipofectamine™ RNAiMAX complexes to form.Meanwhile, HeLa cells were trypsinized and resuspended in medium at afinal concentration of 367 cells/μL. Finally, 450 μL of the cellsuspension were added to each well (final volume 500 μL) and plates wereplaced into the incubator for 24 hours.

RNA Isolation and Analysis, In Vitro

Cells were washed once with 2 mL of PBS, and total RNA was extractedusing RNeasy Mini Kit™ (Qiagen) and eluted in a final volume of 30 μL. 1μg of total RNA was reverse-transcribed using Transcriptor 1^(st) StrandcDNA Kit™ (Roche) and random hexamers following manufacturer'sinstructions. One-thirtieth (0.66 μL) of the resulting cDNA was mixedwith 5 μL of iQ™ Multiplex Powermix (Bio-Rad) together with 3.33 μL ofH₂O and 1 μL of a 3 μM mix containing 2 sets of primers and probesspecific for human genes HPRT-1 (accession number NM_000194) and SFRS9(accession number NM_003769) genes:

Hu HPRT forward primer F517 GACTTTGCTTTCCTTGGTCAGHu HPRT reverse primer  R591 GGCTTATATCCAACACTTCGT GGG Hu HPRT probeP554 Cy5-ATGGTCAAGGTCGCAAG CTTGCTGGT-IBFQ Hu SFRS9 forward primer F569TGTGCAGAAGGATGGAGT Hu SFRS9 reverse primer R712 CTGGTGCTTCTCTCAGGATAHu SFRS9 probe P644 HEX-TGGAATATGCCCTGCG TAAACTGGA-IBFQ

In Vivo Sample Preparation and Injection

DsiRNA was formulated in Invivofectamine™ according to manufacturer'sprotocol (Invitrogen, Carlsbad, Calif.). Briefly, the N/group of miceand body weight of the mice used were determined, then amount of DsiRNAneeded for each group of mice treated was calculated. One ml IVF-oligowas enough for 4 mice of 25 g/mouse at 10 mg/kg dosage. One mg DsiRNAwas added to one ml Invivofectamine™, and mixed at RT for 30 min on arotator. 14 ml of 5% glucose was used to dilute formulated IVF-DsiRNAand was applied to 50 kDa molecular weight cutoff spin concentrators(Amicon). The spin concentrators were spun at 4000 rpm for ˜2 hours at 4C until the volume of IVF-DsiRNA was brought down to less than 1 ml.Recovered IVF-DsiRNA was diluted to one ml with 5% glucose and readiedfor animal injection.

Animal Injection and Tissue Harvesting

Animals were subjected to surgical anesthesia by i.p. injection withKetamine/Xylazine. Each mouse was weighed before injection. FormulatedIVF-DsiRNA was injected i.v. at 100 ul/10 g of body weight. After 24hours, mice were sacrificed by CO₂ inhalation. Tissues for analysis werecollected and placed in tubes containing 2 ml RNAlater™ (Qiagen) androtated at RT for 30 min before incubation at 4° C. overnight. Thetissues were stored subsequently at −80° C. until use.

Tissue RNA Preparation and Quantitation

About 50-100 mg of tissue pieces were homogenized in 1 ml QIAzol™(Qiagen) on Tissue Lyser™ (Qiagen). Then total RNA were isolatedaccording to the manufacturer's protocol. Briefly, 0.2 ml Chloroform(Sigma-Aldrich) was added to the QIAzol™ lysates and mixed vigorously byvortexing. After spinning at 14,000 rpm for 15 min at 4° C., aqueousphase was collected and mixed with 0.5 ml of isopropanol. After anothercentrifugation at 14,000 rpm for 10 min, the RNA pellet was washed oncewith 75% ethanol and briefly dried. The isolated RNA was resuspended in100 μl RNase-Free water, and subjected to clean up with RNeasy™ totalRNA preparation kit (Qiagen) or SV 96 total RNA Isolation System(Promega) according to manufacturer's protocol.

First Strand cDNA Synthesis, In Vivo

1 μg of total RNA was reverse-transcribed using Transcriptor 1^(st)Strand cDNA Kit™ (Roche) and oligo-dT following manufacturer'sinstructions. One-fortieth (0.66 μL) of the resulting cDNA was mixedwith 5 μL of IQ Multiplex Powermix (Bio-Rad) together with 3.33 μL ofH₂O and 1 μL of a 3 μM mix containing 2 sets of primers and probesspecific for mouse genes HPRT-1 (accession number NM_013556) and KRAS(accession number NM_021284) genes:

Mm HPRT forward primer F576 CAAACTTTGCTTTCCCTGGT Mm HPRT reverse primerR664 CAACAAAGTCTGGCCTGTATC Mm HPRT probe P616 Cy5-TGGTTAAGGTTGCAAGCTTGCTGGTG-IBFQ Mm KRAS forward primer F275 CTTTGTGGATGAGTACGACCMm KRAS reverse primer R390 CACTGTACTCCTCTTGACCT Mm KRAS probe P297FAM-ACGATAGAGGACTCCTACA GGAAACAAGT-IBFQ

Quantitative RT-PCR

A CFX96 Real-time System with a C1000 Thermal cycler (Bio-Rad) was usedfor the amplification reactions. PCR conditions were: 95° C. for 3 min;and then cycling at 95° C., 10 sec; 55° C., 1 min for 40 cycles. Eachsample was tested in triplicate. For HPRT Examples, relative HPRT mRNAlevels were normalized to SFRS9 mRNA levels and compared with mRNAlevels obtained in control samples treated with the transfection reagentplus a control mismatch duplex, or untreated. For KRAS examples,relative KRAS mRNA levels were normalized to HPRT-1 mRNA levels andcompared with mRNA levels obtained in control samples from mice treatedwith 5% glucose. Data were analyzed using Bio-Rad CFX Manager version1.0 (in vitro Examples) or 1.5 (in vivo Example) software.

Example 2—Efficacy of DsiRNA Agents Possessing Single StrandedExtensions

DsiRNA agents possessing single stranded extensions were examined forefficacy of sequence-specific target mRNA inhibition. Specifically,KRAS-249M and HPRT-targeting DsiRNA duplexes possessing 5′ singlestranded guide extensions were transfected into HeLa cells at a fixedconcentration of 20 nM and HPRT expression levels were measured 24 hourslater (FIGS. 7 and 9). Transfections were performed in duplicate, andeach duplicate was assayed in triplicate for KRAS-249M and HPRTexpression, respectively, by qPCR.

Under these conditions (0.1 nM duplexes, Lipofectamine™ RNAiMAXtransfection), KRAS-249 gene expression was reduced by about 60-85% byduplexes DNA10PS, RNA10PS, RNA10PS-2′-OME, DNA15PS, RNA15PS, andRNA15PS-2′OME (FIG. 7). By comparison, a duplex without the singlestranded guide extensions reduced KRAS-249 gene expression by about 90%.Thus, the duplexes having single stranded guide extensions were aseffective in silencing KRAS-249 as a duplex without the single strandedguide extensions. All single stranded extended duplexes containedphosphorothioate backbone modifications in the single stranded extensionregion. For duplexes DNA10PS, RNA10PS, RNA10PS-2′-OME, having 10nucleotide single stranded guide extensions, KRAS-249 gene expressionwas reduced about 75-85%. For duplexes DNA10PS, RNA10PS, RNA10PS-2′-OME,having 15 nucleotide single stranded guide extensions, KRAS-249 geneexpression was reduced 60-70%. Generally, the duplexes having the 10nucleotide guide extensions reduced KRAS target gene expression morethan the duplexes having the 15 nucleotide guide extensions, regardlessof the nucleotides present in the 5′ guide extensions. In particular,the silencing activity of duplexes having guide extensions containingdeoxyribonucleotides, was more sensitive to the increased length of 15nucleotides, compared to the duplexes containing ribonucleotides and2′-O-methyl ribonucleotides. Processing of 5′ guide strand extendedduplexes by Dicer, which were used in the experiments targeting geneexpression of KRAS-249, was also shown by in vitro assay (FIG. 10).

Similarly, under the same conditions (0.1 nM duplexes, Lipofectamine™RNAiMAX transfection), HPRT1 gene expression was reduced by about 65-85%by duplexes DNA10PS, RNA10PS, RNA10PS-2′-OME, DNA15PS, RNA15PS, andRNA15PS-2′OME (FIG. 9). By comparison, a duplex without the singlestranded guide extensions reduced HPRT1 gene expression by about 90%.Thus, the duplexes having single stranded guide extensions were aseffective in silencing HPRT1 as a duplex without the single strandedguide extensions. All single stranded extended duplexes containedphosphorothioate backbone modifications in the single stranded extensionregion. For duplexes DNA10PS, RNA10PS, RNA10PS-2′-OME, having 10nucleotide single stranded guide extensions, KRAS-249 gene expressionwas reduced about 80-85%. For duplexes DNA10PS, RNA10PS, RNA10PS-2′-OME,having 15 nucleotide single stranded guide extensions, KRAS-249 geneexpression was reduced 60-80%. Generally, the duplexes having the 10nucleotide guide extensions reduced KRAS target gene expression morethan the duplexes having the 15 nucleotide guide extensions, regardlessof the nucleotides present in the 5′ guide extensions. In particular,the silencing activity of duplexes having guide extensions containingdeoxyribonucleotides or 2′-O-methyl ribonucleotides, was more sensitiveto the increased length of 15 nucleotides, compared to the duplexescontaining ribonucleotides. Processing of 5′ guide strand extendedduplexes by Dicer, which were used in the experiments targeting geneexpression of HPRT1, was also shown by in vitro assay (FIG. 10).

Because the duplex having the single stranded guide extensions were aseffective in silencing KRAS-249 and HPRT1, respectively, as a duplexwithout the single stranded guide extensions, this discovery allows forthe modification of DsiRNA agents with single stranded guide extensionswithout loss of efficacy.

Example 3—Efficacy of DsiRNA Agents Possessing Single StrandedExtensions in Combination with a Short Oligonucleotide Complementary tothe Single Stranded Extension

DsiRNA agents possessing single stranded extensions were examined forefficacy of sequence-specific target mRNA inhibition in combination witha short oligo complementary to the single stranded extension.Specifically, KRAS-249M and HPRT-targeting DsiRNA duplexes possessing 15nucleotide long 5′ single stranded guide extensions including a 15nucleotide discontinuous complement were transfected into HeLa cells ata fixed concentration of 20 nM and HPRT expression levels were measured24 hours later (FIGS. 12 and 13). Transfections were performed induplicate, and each duplicate was assayed in triplicate for KRAS-249Mand HPRT expression, respectively, by qPCR.

Under these conditions (0.1 nM duplexes, Lipofectamine™ RNAiMAXtransfection), KRAS-249 gene expression was reduced by about 15-60% byduplexes DNA15PS (1301+1340), RNA15PS (1301+1341), RNA15PS-2′-OME(1301+1342) in the presence of discontinuous complements RNA15,PS-RNA15, PS-DNA15, PS-2′OMe-RNA15, and 2′OMe-RNA15 (FIG. 12). A duplexwithout the single stranded guide extensions reduced KRAS-249 geneexpression by about 85%. All single stranded extended duplexes containedphosphorothioate backbone modifications in the single stranded extensionregion. Generally, the duplexes having ribonucleotide or 2′-O-methylribonucleotide guide extensions reduced KRAS target gene expression morethan the duplexes having deoxyribonucleotide guide extensions,regardless of the discontinuous complement present. For duplexes DNA15PS(1301+1340), RNA15PS (1301+1341), RNA15PS-2′-OME (1301+1342), thereductions in gene expression were comparable with or without the2′OMe-RNA15 discontinuous complement.

Similarly, under the same conditions (0.1 nM duplexes, Lipofectamine™RNAiMAX transfection), HPRT1 gene expression was reduced by about 30-85%by duplexes DNA15PS (1001+1353), RNA15PS (1001+1354), and RNA15PS-2′OME(1001+1355) in the presence of discontinuous complements RNA15,PS-RNA15, PS-DNA15, PS-2′OMe-RNA15, and 2′OMe-RNA15 (FIG. 13). A duplexwithout the single stranded guide extensions reduced HPRT1 geneexpression by about 90%. All single stranded extended duplexes containedphosphorothioate backbone modifications in the single stranded extensionregion. Generally, the duplexes having ribonucleotide or 2′-O-methylribonucleotide guide extensions reduced KRAS target gene expression morethan the duplexes having deoxyribonucleotide guide extensions,regardless of the discontinuous complement present. Duplexes RNA15PS(1301+1341) and RNA15PS-2′-OME (1301+1342), showed enhanced reduction ingene expression in the presence of discontinuous complements RNA15,PS-RNA15, PS-2′OMe-RNA15, 2′OMe-RNA15, compared to the same duplexesRNA15PS (1301+1341) and RNA15PS-2′-OME (1301+1342) without anydiscontinuous complement.

Example 4—In Vivo Efficacy of DsiRNA Agents

DsiRNA agents possessing DNA duplex extensions were examined for in vivoefficacy of sequence-specific target mRNA inhibition either in a singledose protocol or in a repeated dose protocol (e.g., single 10 mg/kginjection in invivoFectamine). Expression of KRAS in liver, kidney,spleen and lymph node tissues was measured 24 hours post-injection, withreal-time PCR (RT-PCR) performed in triplicate to assess KRASexpression. Under these conditions, single stranded guide extendedDsiRNA agents exhibited statistically significant levels of KRAS targetgene inhibition in all tissues examined. KRAS percent inhibition levelsin such single stranded guide extension DsiRNA treated tissues were:liver (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,99%, or 100%), spleen (10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,96%, 97%, 98%, 99%, or 100%),), kidney (1910%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%),) and lymph nodes (10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or100%)). Thus, the in vivo efficacy of the extended DsiRNAs of theinstant invention was demonstrated across many tissue types.

Further demonstration of the capability of the extended Dicer substrateagents of the invention to reduce gene expression of specific targetgenes in vivo was performed via administration of the DsiRNAs of theinvention to mice or other mammalian subjects, either systemically(e.g., by i.v. or i.p. injection) or via direct injection of a tissue(e.g., injection of the eye, spinal cord/brain/CNS, etc.). Measurementof additional target RNA levels were performed upon target cells (e.g.,RNA levels in liver and/or kidney cells were assayed following injectionof mice; eye cells were assayed following ophthalmic injection ofsubjects; or spinal cord/brain/CNS cells were assayed following directinjection of same of subjects) by standard methods (e.g., Trizol®preparation (guanidinium thiocyanate-phenol-chloroform) followed byqRT-PCR).

In any such further in vivo experiments, an extended Dicer substrateagent of the invention (e.g., a guide 5′extended or passenger 3′extendedDsiRNA) can be deemed to be an effective in vivo agent if astatistically significant reduction in RNA levels was observed whenadministering an extended Dicer substrate agent of the invention, ascompared to an appropriate control (e.g., a vehicle alone control, arandomized duplex control, a duplex directed to a different target RNAcontrol, etc.). Generally, if the p-value (e.g., generated via 1 tailed,unpaired T-test) assigned to such comparison was less than 0.05, anextended Dicer substrate agent (e.g., guide 5′extended or passenger3′extended DsiRNA agent) of the invention was deemed to be an effectiveRNA interference agent. Alternatively, the p-value threshold below whichto classify an extended Dicer substrate agent of the invention as aneffective RNA interference agent can be set, e.g. at 0.01, 0.001, etc.,in order to provide more stringent filtering, identify more robustdifferences, and/or adjust for multiple hypothesis testing, etc.Absolute activity level limits can also be set to distinguish betweeneffective and non-effective extended Dicer substrate agents. Forexample, in certain embodiments, an effective extended Dicer substrateagent of the invention was one that not only shows a statisticallysignificant reduction of target RNA levels in vivo but also exerts,e.g., at least an approximately 10% reduction, approximately 15%reduction, at least approximately 20% reduction, approximately 25%reduction, approximately 30% reduction, etc. in target RNA levels in thetissue or cell that was examined, as compared to an appropriate control.Further in vivo efficacy testing of the extended Dicer substrate agents(e.g., guide 5′extended and passenger 3′extended DsiRNA agents) of theinvention was thereby performed.

DsiRNA agents possessing single stranded extensions (FIGS. 14 and 15)effectively inhibited the sequence-specific target KRAS mRNA expressionin vivo in liver, spleen, and kidney. In liver, the 5′ passengerextended DsiRNA agents 1371 (PS 3M) and 1339 (PS 10M) showed inhibitionof KRAS mRNA expression as compared to DsiRNA agents without the 5′passenger extensions K249M and 1370 (3M), when normalized to glucoseonly control (FIGS. 16-18). The inhibition of KRAS mRNA expression bythe DsiRNA agents was at least 75-90% in liver of animals injected withthe 5′ passenger extended DsiRNA agents 1371 (PS 3M) and 1339 (PS 10M).The amount of inhibition of the 5′ passenger extended DsiRNA agents 1371(PS 3M) and 1339 (PS 10M) in liver was comparable to that of DsiRNAagents without the 5′ passenger extensions K249M and 1370 (3M), whichwas significant compared to the negative glucose control.

In spleen, the 5′ passenger extended DsiRNA agents 1371 (PS 3M) and 1339(PS 10M) also showed inhibition of KRAS mRNA expression as compared toDsiRNA agents without the 5′ passenger extensions K249M and 1370 (3M),when normalized to glucose only control (FIGS. 19-21). The inhibition ofKRAS mRNA expression by the DsiRNA agents was at least 90-95% in spleenof animals injected with the 5′ passenger extended DsiRNA agents 1371(PS 3M) and 1339 (PS 10M). The amount of inhibition of the 5′ passengerextended DsiRNA agents 1371 (PS 3M) and 1339 (PS 10M) in spleen wascomparable to that of DsiRNA agents without the 5′ passenger extensionsK249M and 1370 (3M), which was significant compared to the negativeglucose control.

In kidney, the 5′ passenger extended DsiRNA agents 1371 (PS 3M) and 1339(PS 10M) showed inhibition of KRAS mRNA expression as compared to DsiRNAagents without the 5′ passenger extensions K249M and 1370 (3M), whennormalized to glucose only control (FIGS. 22-24). The inhibition of KRASmRNA expression by the DsiRNA agents was at least 20-40% in kidney ofanimals injected with the 5′ passenger extended DsiRNA agents 1371 (PS3M) and 1339 (PS 10M). Nevertheless, the amount of inhibition of the 5′passenger extended DsiRNA agents 1371 (PS 3M) and 1339 (PS 10M) wascomparable to that of DsiRNA agents without the 5′ passenger extensionsK249M and 1370 (3M). In these experiments, a DsiRNA agent without the 5′passenger extension M97M and not sequence specific to KRAS was used as apositive control.

Because the DsiRNA agents having a single stranded guide extension wereas effective in silencing KRAS in vivo, as DsiRNA agents without thesingle stranded guide extension, this discovery allows for themodification of DsiRNA agents with single stranded guide extensionswithout loss of efficacy in vivo.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present invention teaches oneskilled in the art to test various combinations and/or substitutions ofchemical modifications described herein toward generating nucleic acidconstructs with improved activity for mediating RNAi activity. Suchimproved activity can comprise improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying DsiRNA moleculeswith improved RNAi activity.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

1.-69. (canceled)
 70. An isolated double stranded nucleic acidcomprising: a first oligonucleotide strand having a 5′ terminus and a 3′terminus and a second oligonucleotide strand having a 5′ terminus and a3′ terminus, wherein each said 5′ terminus comprises a 5′ terminalnucleotide and each said 3′ terminus comprises a 3′ terminal nucleotide;and an extended loop at the 3′ terminus of said first strand or the 5′terminus of said second strand; wherein said first strand is 35-60nucleotide residues in length; wherein said second strand is 19-39nucleotide residues in length; wherein from 1-30 consecutivenucleotides, including the 5′ terminal nucleotide, of said first strandare unpaired with the 3′ terminus of said second strand, thereby forminga 1-30 nucleotide single stranded 5′ overhang on said first strand;wherein said first strand is annealed to said second strand to form aduplex region of 18-30 base pairs; and wherein said second strand issufficiently complementary to a target RNA along at least 19ribonucleotides of said second strand length to reduce target geneexpression when said double stranded nucleic acid is introduced into amammalian cell.
 71. The isolated double stranded nucleic acid of claim70, wherein at least one nucleotide of said first strand between andincluding said first strand positions 24 to the 3′ terminal nucleotideresidue of said first strand is a deoxyribonucleotide that base pairswith said second strand.
 72. The isolated double stranded nucleic acidof claim 70, wherein at least 10 consecutive nucleotides and at most 15consecutive nucleotides, including the 5′ terminal nucleotide of saidfirst strand are unpaired with the 3′ terminus of said second strand,thereby forming a 10-15 nucleotide single stranded 5′ overhang.
 73. Theisolated double stranded nucleic acid of claim 70, wherein at least onenucleotide of said second strand between and including second strandnucleotides corresponding to and thus base paired with first strandpositions 24 to the 3′ terminal nucleotide residue of said first strandis a ribonucleotide.
 74. The isolated double stranded nucleic acid ofclaim 70, wherein said duplex between said first and second strandscomprises a fully duplexed region having no unpaired bases.
 75. Theisolated double stranded nucleic acid of claim 70, wherein said duplexbetween said first and second strands comprises an unpaired base pairselected from the group consisting of: 1 unpaired base pair; 2 unpairedbase pairs, 3 unpaired base pairs, 4 unpaired base pairs, and 5 unpairedbase pairs.
 76. The isolated double stranded nucleic acid of claim 75,wherein said unpaired base pairs are consecutive or non-consecutive. 77.The isolated double stranded nucleic acid of claim 70, wherein saidfirst strand is up to 60 nucleotides in length, and the nucleotides ofsaid first strand 3′ to position 23 of said first strand comprisesdeoxyribonucleotides selected from the group consisting of: two, three,four, five, and six nucleotide residues of positions 24 to the 3′terminal nucleotide residue of said first strand aredeoxyribonucleotides.
 78. The isolated double stranded nucleic acid ofclaim 77, wherein said deoxyribonucleotides are consecutivedeoxyribonucleotides.
 79. The isolated double stranded nucleic acid ofclaim 70, wherein two or more consecutive nucleotide residues ofpositions 24 to 30 of said first strand are deoxyribonucleotides thatbase pair with nucleotides of said second strand.
 80. The isolateddouble stranded nucleic acid of claim 79, wherein said first strand isup to 60 nucleotides in length and comprises a pair ofdeoxyribonucleotides selected from the group consisting of: positions 24and 25, positions 25 and 26, positions 26 and 27, positions 27 and 28,positions 28 and 29, and positions 29 and 30, wherein said first pair ofdeoxyribonucleotides is base paired with a corresponding pair ofnucleotides of said second strand.
 81. The isolated double strandednucleic acid of claim 70, wherein starting from the 5′ terminalnucleotide (position 1) positions 1 to 28 of said first strand compriseat least 8 ribonucleotides.
 82. The isolated double stranded nucleicacid of claim 70, wherein said first strand 5′ overhang is selected fromthe group consisting of: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29 and 30 nucleotides in length.
 83. Theisolated double stranded nucleic acid of claim 70, wherein said firststrand 5′ overhang comprises a ribonucleotide or deoxyribonucleotide.84. The isolated double stranded nucleic acid of claim 83, wherein allnucleotides of said first strand 5′ overhang are deoxyribonucleotides.85. The isolated double stranded nucleic acid of claim 82, wherein saidnucleotides of said first strand 5′ overhang comprise a phosphatebackbone modification.
 86. The isolated double stranded nucleic acid ofclaim 85, wherein said phosphate backbone modification is selected fromthe group consisting of a phosphonate, a phosphorothioate, aphosphotriester, a methylphosphonate, a locked nucleic acid, amorpholino and a bicyclic furanose analog.
 87. The isolated doublestranded nucleic acid of claim 86, wherein the 5′ terminal nucleotide ofsaid first strand is a ribonucleotide.
 88. The isolated double strandednucleic acid of claim 70, wherein said nucleotides of said first strand5′ overhang comprise a modified nucleotide.
 89. The isolated doublestranded nucleic acid of claim 88, wherein said modified nucleotideresidue is selected from the group consisting of 2′-O-methyl,2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge,4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino and 2′-O—(N-methylcarbamate).90. The isolated double stranded nucleic acid of claim 89, wherein saidmodified nucleotide of said first strand 5′ overhang is a 2′-O-methylribonucleotide.
 91. The isolated double stranded nucleic acid of claim70, wherein a nucleotide of said second or first oligonucleotide strandis substituted with a modified nucleotide that directs the orientationof Dicer cleavage.
 92. The isolated double stranded nucleic acid ofclaim 70, wherein the first strand has a nucleotide sequence that is atleast 80%, 90%, 95% or 100% complementary to the second strandnucleotide sequence.
 93. The isolated double stranded nucleic acid ofclaim 70, wherein said target RNA is KRAS.
 94. A pharmaceuticalcomposition for reducing expression of a target gene in a cell of asubject comprising an isolated double stranded nucleic acid as claimedin claim 70 in an amount effective to reduce expression of a target genein a cell in comparison to a reference dsRNA and a pharmaceuticallyacceptable carrier.
 95. A method of synthesizing a double strandednucleic acid as claimed in claim 88, comprising chemically orenzymatically synthesizing said double stranded nucleic acid.
 96. Theisolated double stranded nucleic acid of claim 70, wherein said secondstrand is 26-36 nucleotide residues in length.
 97. The isolated doublestranded nucleic acid of claim 70, wherein the second strand startingfrom the 3′ terminal nucleotide, comprises at least 8 ribonucleotides inthe positions paired with positions 1-23 of said first strand to form aduplex.
 98. The isolated double stranded nucleic acid of claim 70,wherein the double-stranded nucleic acid is conjugated to a non-nucleicacid moiety.
 99. The isolated double stranded nucleic acid of claim ofclaim 98, wherein the non-nucleic acid moiety is a peptide or an organiccompound.
 100. The isolated double stranded nucleic acid of claim 70,wherein the extended loop is a tetraloop.
 101. The isolated doublestranded nucleic acid of claim 100, wherein the extended loop links thefirst and second strand.